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Sample records for cryptic splice sites

  1. A novel mutation in the β-spectrin gene causes the activation of a cryptic 5'-splice site and the creation of a de novo 3'-splice site.

    Science.gov (United States)

    Salas, Pilar Carrasco; Rosales, José Miguel Lezana; Milla, Carmen Palma; Montiel, Javier López; Siles, Juan López

    2015-01-01

    The analysis of genes involved in hereditary spherocytosis, by next-generation sequencing in two patients with clinical diagnosis of the disease, showed the presence of the c.1795+1G>A mutation in the SPTB gene. cDNA amplification then revealed the occurrence of a consequent aberrant mRNA isoform produced from the activation of a cryptic 5'-splice site and the creation of a newly 3'-splice site. The mechanisms by which these two splice sites are used as a result of the same mutation should be analyzed in depth in further studies.

  2. Biased exon/intron distribution of cryptic and de novo 3' splice sites.

    Science.gov (United States)

    Královicová, Jana; Christensen, Mikkel B; Vorechovský, Igor

    2005-01-01

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

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

    Science.gov (United States)

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

    2013-03-01

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

  4. Complete androgen insensitivity syndrome caused by a novel splice donor site mutation and activation of a cryptic splice donor site in the androgen receptor gene.

    Science.gov (United States)

    Infante, Joana B; Alvelos, Maria I; Bastos, Margarida; Carrilho, Francisco; Lemos, Manuel C

    2016-01-01

    The androgen insensitivity syndrome is an X-linked recessive genetic disorder characterized by resistance to the actions of androgens in an individual with a male karyotype. We evaluated a 34-year-old female with primary amenorrhea and a 46,XY karyotype, with normal secondary sex characteristics, absence of uterus and ovaries, intra-abdominal testis, and elevated testosterone levels. Sequence analysis of the androgen receptor (AR) gene revealed a novel splice donor site mutation in intron 4 (c.2173+2T>C). RT-PCR analysis showed that this mutation resulted in the activation of a cryptic splice donor site located in the second half of exon 4 and in the synthesis of a shorter mRNA transcript and an in-frame deletion of 41 amino acids. This novel mutation associated with a rare mechanism of abnormal splicing further expands the spectrum of mutations associated with the androgen insensitivity syndrome and may contribute to the understanding of the molecular mechanisms involved in splicing defects.

  5. Two novel splicing mutations in the SLC45A2 gene cause Oculocutaneous Albinism Type IV by unmasking cryptic splice sites.

    Science.gov (United States)

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

    2015-09-01

    Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type IV (OCA4) is one of the four commonly recognized forms of albinism, and is determined by mutation in the SLC45A2 gene. Here, we investigated the genetic basis of OCA4 in an Italian child. The mutational screening of the SLC45A2 gene identified two novel potentially pathogenic splicing mutations: a synonymous transition (c.888G>A) involving the last nucleotide of exon 3 and a single-nucleotide insertion (c.1156+2dupT) within the consensus sequence of the donor splice site of intron 5. As computer-assisted analysis for mutant splice-site prediction was not conclusive, we investigated the effects on pre-mRNA splicing of these two variants by using an in vitro minigene approach. Production of mutant transcripts in HeLa cells demonstrated that both mutations cause the almost complete abolishment of the physiologic donor splice site, with the concomitant unmasking of cryptic donor splice sites. To our knowledge, this work represents the first in-depth molecular characterization of splicing defects in a OCA4 patient.

  6. A novel mutation in the β-spectrin gene causes the activation of a cryptic 5′-splice site and the creation of a de novo 3′-splice site

    Science.gov (United States)

    Salas, Pilar Carrasco; Rosales, José Miguel Lezana; Milla, Carmen Palma; Montiel, Javier López; Siles, Juan López

    2015-01-01

    The analysis of genes involved in hereditary spherocytosis, by next-generation sequencing in two patients with clinical diagnosis of the disease, showed the presence of the c.1795+1G>A mutation in the SPTB gene. cDNA amplification then revealed the occurrence of a consequent aberrant mRNA isoform produced from the activation of a cryptic 5′-splice site and the creation of a newly 3′-splice site. The mechanisms by which these two splice sites are used as a result of the same mutation should be analyzed in depth in further studies. PMID:27081538

  7. Activation of a cryptic splice site in the mitochondrial elongation factor GFM1 causes combined OXPHOS deficiency☆

    Science.gov (United States)

    Simon, Mariella T.; Ng, Bobby G.; Friederich, Marisa W.; Wang, Raymond Y.; Boyer, Monica; Kircher, Martin; Collard, Renata; Buckingham, Kati J.; Chang, Richard; Shendure, Jay; Nickerson, Deborah A.; Bamshad, Michael J.; Van Hove, Johan L.K.; Freeze, Hudson H.; Abdenur, Jose E.

    2017-01-01

    We report the clinical, biochemical, and molecular findings in two brothers with encephalopathy and multi-systemic disease. Abnormal transferrin glycoforms were suggestive of a type I congenital disorder of glycosylation (CDG). While exome sequencing was negative for CDG related candidate genes, the testing revealed compound heterozygous mutations in the mitochondrial elongation factor G gene (GFM1). One of the mutations had been reported previously while the second, novel variant was found deep in intron 6, activating a cryptic splice site. Functional studies demonstrated decreased GFM1 protein levels, suggested disrupted assembly of mitochondrial complexes III and V and decreased activities of mitochondrial complexes I and IV, all indicating combined OXPHOS deficiency. PMID:28216230

  8. From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: a Pipeline for the Development of Antisense Oligonucleotides

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    Atze J Bergsma

    2016-01-01

    Full Text Available While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1993-08-01

    Galactosemia affects 1/84,000 in Sweden and is manifested in infancy when the child is exposed to galactose in the diet. If untreated there is a risk of severe early symptoms and, even with a lactose-free diet, late symptoms such as mental retardation and ovarial dysfunction may develop. In classical galactosemia, galactose-1-phosphate uridyltransferase (GALT) (EC 2.7.7.12) is defective and the normal cDNA sequence of this enzyme has been characterized. Recently eight mutations leading to galactosemia were published. Heparinized venous blood was drawn from a patient with classical galactosemia. In the cDNA from the patient examined, an insertion of 54 bp was found at position 1087. Amplification of the relevant genomic region of the patient's DNA was performed. Exon-intron boundaries and intronic sequences thus determined revealed that the 54-bp insertion was located immediately downstream of exon 10. It was further found that the patient was heterozygous for a point mutation, changing a C to a T (in 5 of 9 clones) at the second base in the intron downstream of the insertion. This alteration creates a sequence which, as well as the ordinary splice site, differs in only two positions from the consensus sequence. It was found that the mutation occurred in only one of the 20 alleles from galactosemic patients and in none of the 200 alleles from normal controls. The mutation is inherited from the mother, who also was found to express the 54-bp-long insertion at the mRNA level. Sequences from the 5[prime] end of the coding region were determined after genomic amplification, revealing a sequence identical to that reported. The mutation on the paternal allele has not been identified. 9 refs., 1 fig.

  10. Quantitative analysis of cryptic splicing associated with TDP-43 depletion.

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    Humphrey, Jack; Emmett, Warren; Fratta, Pietro; Isaacs, Adrian M; Plagnol, Vincent

    2017-05-26

    Reliable exon recognition is key to the splicing of pre-mRNAs into mature mRNAs. TDP-43 is an RNA-binding protein whose nuclear loss and cytoplasmic aggregation are a hallmark pathology in amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). TDP-43 depletion causes the aberrant inclusion of cryptic exons into a range of transcripts, but their extent, relevance to disease pathogenesis and whether they are caused by other RNA-binding proteins implicated in ALS/FTD are unknown. We developed an analysis pipeline to discover and quantify cryptic exon inclusion and applied it to publicly available human and murine RNA-sequencing data. We detected widespread cryptic splicing in TDP-43 depletion datasets but almost none in another ALS/FTD-linked protein FUS. Sequence motif and iCLIP analysis of cryptic exons demonstrated that they are bound by TDP-43. Unlike the cryptic exons seen in hnRNP C depletion, those repressed by TDP-43 cannot be linked to transposable elements. Cryptic exons are poorly conserved and inclusion overwhelmingly leads to nonsense-mediated decay of the host transcript, with reduced transcript levels observed in differential expression analysis. RNA-protein interaction data on 73 different RNA-binding proteins showed that, in addition to TDP-43, 7 specifically bind TDP-43 linked cryptic exons. This suggests that TDP-43 competes with other splicing factors for binding to cryptic exons and can repress cryptic exon inclusion. Our quantitative analysis pipeline confirms the presence of cryptic exons during the depletion of TDP-43 but not FUS providing new insight into to RNA-processing dysfunction as a cause or consequence in ALS/FTD.

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

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

    1994-09-01

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

  12. Extensive cryptic splicing upon loss of RBM17 and TDP43 in neurodegeneration models.

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    Tan, Qiumin; Yalamanchili, Hari Krishna; Park, Jeehye; De Maio, Antonia; Lu, Hsiang-Chih; Wan, Ying-Wooi; White, Joshua J; Bondar, Vitaliy V; Sayegh, Layal S; Liu, Xiuyun; Gao, Yan; Sillitoe, Roy V; Orr, Harry T; Liu, Zhandong; Zoghbi, Huda Y

    2016-12-01

    Splicing regulation is an important step of post-transcriptional gene regulation. It is a highly dynamic process orchestrated by RNA-binding proteins (RBPs). RBP dysfunction and global splicing dysregulation have been implicated in many human diseases, but the in vivo functions of most RBPs and the splicing outcome upon their loss remain largely unexplored. Here we report that constitutive deletion of Rbm17, which encodes an RBP with a putative role in splicing, causes early embryonic lethality in mice and that its loss in Purkinje neurons leads to rapid degeneration. Transcriptome profiling of Rbm17-deficient and control neurons and subsequent splicing analyses using CrypSplice, a new computational method that we developed, revealed that more than half of RBM17-dependent splicing changes are cryptic. Importantly, RBM17 represses cryptic splicing of genes that likely contribute to motor coordination and cell survival. This finding prompted us to re-analyze published datasets from a recent report on TDP-43, an RBP implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as it was demonstrated that TDP-43 represses cryptic exon splicing to promote cell survival. We uncovered a large number of TDP-43-dependent splicing defects that were not previously discovered, revealing that TDP-43 extensively regulates cryptic splicing. Moreover, we found a significant overlap in genes that undergo both RBM17- and TDP-43-dependent cryptic splicing repression, many of which are associated with survival. We propose that repression of cryptic splicing by RBPs is critical for neuronal health and survival. CrypSplice is available at www.liuzlab.org/CrypSplice. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  13. A cryptic BAP1 splice mutation in a family with uveal and cutaneous melanoma, and paraganglioma

    DEFF Research Database (Denmark)

    Wadt, K.; Choi, J.; Chung, J.Y.;

    2012-01-01

    as paraganglioma, breast cancer, and suspected mesothelioma cases in the family. Bioinformatic analysis and splicing assays demonstrated that this mutation creates a strong cryptic splice donor, resulting in aberrant splicing and a truncating frameshift of the BAP1 transcript. Somatic loss of the wild-type allele...

  14. Gene trap mutagenesis of hnRNP A2/B1: a cryptic 3' splice site in the neomycin resistance gene allows continued expression of the disrupted cellular gene

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    DeGregori James V

    2003-01-01

    Full Text Available Abstract Background Tagged sequence mutagenesis is a process for constructing libraries of sequenced insertion mutations in embryonic stem cells that can be transmitted into the mouse germline. To better predict the functional consequences of gene entrapment on cellular gene expression, the present study characterized the effects of a U3Neo gene trap retrovirus inserted into an intron of the hnRNP A2/B1 gene. The mutation was selected for analysis because it occurred in a highly expressed gene and yet did not produce obvious phenotypes following germline transmission. Results Sequences flanking the integrated gene trap vector in 1B4 cells were used to isolate a full-length cDNA whose predicted amino acid sequence is identical to the human A2 protein at all but one of 341 amino acid residues. hnRNP A2/B1 transcripts extending into the provirus utilize a cryptic 3' splice site located 28 nucleotides downstream of the neomycin phosphotransferase start codon. The inserted Neo sequence and proviral poly(A site function as an 3' terminal exon that is utilized to produce hnRNP A2/B1-Neo fusion transcripts, or skipped to produce wild-type hnRNP A2/B1 transcripts. This results in only a modest disruption of hnRNPA2/B1 gene expression. Conclusions Expression of the occupied hnRNP A2/B1 gene and utilization of the viral poly(A site are consistent with an exon definition model of pre-mRNA splicing. These results reveal a mechanism by which U3 gene trap vectors can be expressed without disrupting cellular gene expression, thus suggesting ways to improve these vectors for gene trap mutagenesis.

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

    Science.gov (United States)

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

    1993-01-01

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

  16. Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic syndromes.

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    Dolatshad, H; Pellagatti, A; Liberante, F G; Llorian, M; Repapi, E; Steeples, V; Roy, S; Scifo, L; Armstrong, R N; Shaw, J; Yip, B H; Killick, S; Kušec, R; Taylor, S; Mills, K I; Savage, K I; Smith, C W J; Boultwood, J

    2016-12-01

    The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3' splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3' splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.

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

    DEFF Research Database (Denmark)

    Hebsgaard, Stefan M.; Korning, Peter G.; Tolstrup, Niels

    1996-01-01

    splice sites normally haunting splice site prediction. An analysis of the errors made by the networks in the first step of the method revealed a previously unknown feature, a frequent T-tract prolongation containing cryptic acceptor sites in the 5'end of exons. The method presented here has been compared...

  18. Oriented scanning is the leading mechanism underlying 5' splice site selection in mammals.

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    Keren Borensztajn

    2006-09-01

    Full Text Available Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5' splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7 a 37-bp VNTR minisatellite whose first element spans the exon7-IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5' pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5' splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5' splice site, rather than repressing the 3' following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5' pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 5' splice site follows a scanning-type mechanism, precluding competition with other functional 5' pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 5' pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type-independent 5'-3'-oriented scanning process for accurate recognition of the authentic 5' splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 5' splice site selection.

  19. Characterization of cryptic splicing in germline PTEN intronic variants in Cowden syndrome.

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    Chen, Hannah Jinlian; Romigh, Todd; Sesock, Kaitlin; Eng, Charis

    2017-10-01

    Germline mutations in the tumor-suppressor gene PTEN predispose to subsets of Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome, and autism. Evidence-based classification of PTEN variants as either deleterious or benign is urgently needed for accurate molecular diagnosis and gene-informed genetic counseling. We studied 34 different germline PTEN intronic variants from 61 CS patients, characterized their PTEN mRNA processing, and analyzed PTEN expression and downstream readouts of P-AKT and P-ERK1/2. While we found that many mutations near splice junctions result in exon skipping, we also identified the presence of cryptic splicing that resulted in premature termination or a shift in isoform usage. PTEN protein expression is significantly lower in the group with splicing changes while P-AKT, but not P-ERK1/2, is significantly increased. Our observations of these PTEN intronic variants should contribute to the determination of pathogenicity of PTEN intronic variants and aid in genetic counseling. © 2017 The Authors. Human Mutation published by Wiley Periodicals, Inc.

  20. GC content around splice sites affects splicing through pre-mRNA secondary structures

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    Chen Liang

    2011-01-01

    Full Text Available Abstract Background Alternative splicing increases protein diversity by generating multiple transcript isoforms from a single gene through different combinations of exons or through different selections of splice sites. It has been reported that RNA secondary structures are involved in alternative splicing. Here we perform a genomic study of RNA secondary structures around splice sites in humans (Homo sapiens, mice (Mus musculus, fruit flies (Drosophila melanogaster, and nematodes (Caenorhabditis elegans to further investigate this phenomenon. Results We observe that GC content around splice sites is closely associated with the splice site usage in multiple species. RNA secondary structure is the possible explanation, because the structural stability difference among alternative splice sites, constitutive splice sites, and skipped splice sites can be explained by the GC content difference. Alternative splice sites tend to be GC-enriched and exhibit more stable RNA secondary structures in all of the considered species. In humans and mice, splice sites of first exons and long exons tend to be GC-enriched and hence form more stable structures, indicating the special role of RNA secondary structures in promoter proximal splicing events and the splicing of long exons. In addition, GC-enriched exon-intron junctions tend to be overrepresented in tissue-specific alternative splice sites, indicating the functional consequence of the GC effect. Compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched. We also found that the GC-content effect is much stronger than the nucleotide-order effect to form stable secondary structures. Conclusion All of these results indicate that GC content is related to splice site usage and it may mediate the splicing process through RNA secondary structures.

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

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    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

    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

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

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    Mireia Mora

    Full Text Available 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.

  3. CYP17A1 intron mutation causing cryptic splicing in 17α-hydroxylase deficiency.

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    Daw-Yang Hwang

    Full Text Available 17α-Hydroxylase/17, 20-lyase deficiency (17OHD is an autosomal recessive disease causing congenital adrenal hyperplasia and a rare cause of hypertension with hypokalemia. The CYP17A1 gene mutation leads to 17OHD and its clinical features. We described an 18 y/o female with clinical features of 17α-hydroxylase/17, 20-lyase deficiency and characterized the functional consequences of an intronic CYP17A1 mutation. The coding regions and flanking intronic bases of the CYP17A1 gene were amplified by PCR and sequenced. The patient is a compound heterozygote for the previously described p.R358X and IVS1 +2T>C mutations. A first intron splice donor site mutation was re-created in minigene and full-length expression vectors. Pre-mRNA splicing of the variant CYP17A1 intron was studied in transfected cells and in a transformed lymphoblastoid cell line. When the full-length CYP17A1 gene and minigene containing the intronic mutation was expressed in transfected cells, the majority (>90% of mRNA transcripts were incorrectly spliced. Only the p.R358X transcript was detected in the EBV-transformed lymphoblastoid cell line. The IVS1 +2T>C mutation abolished most 17α-hydroxylase/17, 20-lyase enzyme activity by aberrant mRNA splicing to an intronic pseudo-exon, causing a frame shift and early termination.

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

    Science.gov (United States)

    Kralovicova, Jana

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Rogozin Igor B

    2006-12-01

    Full Text Available Abstract Background The signals that determine the specificity and efficiency of splicing are multiple and complex, and are not fully understood. Among other factors, the relative contributions of different mechanisms appear to depend on intron size inasmuch as long introns might hinder the activity of the spliceosome through interference with the proper positioning of the intron-exon junctions. Indeed, it has been shown that the information content of splice sites positively correlates with intron length in the nematode, Drosophila, and fungi. We explored the connections between the length of vertebrate introns, the strength of splice sites, exonic splicing signals, and evolution of flanking exons. Results A compensatory relationship is shown to exist between different types of signals, namely, the splice sites and the exonic splicing enhancers (ESEs. In the range of relatively short introns (approximately, Conclusion Several weak but statistically significant correlations were observed between vertebrate intron length, splice site strength, and potential exonic splicing signals. Taken together, these findings attest to a compensatory relationship between splice sites and exonic splicing signals, depending on intron length.

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

    Science.gov (United States)

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

    1999-06-15

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

  7. DNA splice site sequences clustering method for conservativeness analysis

    Institute of Scientific and Technical Information of China (English)

    Quanwei Zhang; Qinke Peng; Tao Xu

    2009-01-01

    DNA sequences that are near to splice sites have remarkable conservativeness,and many researchers have contributed to the prediction of splice site.In order to mine the underlying biological knowledge,we analyze the conservativeness of DNA splice site adjacent sequences by clustering.Firstly,we propose a kind of DNA splice site sequences clustering method which is based on DBSCAN,and use four kinds of dissimilarity calculating methods.Then,we analyze the conservative feature of the clustering results and the experimental data set.

  8. Computational analysis of splicing errors and mutations in human transcripts

    Directory of Open Access Journals (Sweden)

    Gelfand Mikhail S

    2008-01-01

    Full Text Available Abstract Background Most retained introns found in human cDNAs generated by high-throughput sequencing projects seem to result from underspliced transcripts, and thus they capture intermediate steps of pre-mRNA splicing. On the other hand, mutations in splice sites cause exon skipping of the respective exon or activation of pre-existing cryptic sites. Both types of events reflect properties of the splicing mechanism. Results The retained introns were significantly shorter than constitutive ones, and skipped exons are shorter than exons with cryptic sites. Both donor and acceptor splice sites of retained introns were weaker than splice sites of constitutive introns. The authentic acceptor sites affected by mutations were significantly weaker in exons with activated cryptic sites than in skipped exons. The distance from a mutated splice site to the nearest equivalent site is significantly shorter in cases of activated cryptic sites compared to exon skipping events. The prevalence of retained introns within genes monotonically increased in the 5'-to-3' direction (more retained introns close to the 3'-end, consistent with the model of co-transcriptional splicing. The density of exonic splicing enhancers was higher, and the density of exonic splicing silencers lower in retained introns compared to constitutive ones and in exons with cryptic sites compared to skipped exons. Conclusion Thus the analysis of retained introns in human cDNA, exons skipped due to mutations in splice sites and exons with cryptic sites produced results consistent with the intron definition mechanism of splicing of short introns, co-transcriptional splicing, dependence of splicing efficiency on the splice site strength and the density of candidate exonic splicing enhancers and silencers. These results are consistent with other, recently published analyses.

  9. Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

    Directory of Open Access Journals (Sweden)

    Elela Sherif

    2006-01-01

    Full Text Available Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

  10. [Statistical analysis of DNA sequences nearby splicing sites].

    Science.gov (United States)

    Korzinov, O M; Astakhova, T V; Vlasov, P K; Roĭtberg, M A

    2008-01-01

    Recognition of coding regions within eukaryotic genomes is one of oldest but yet not solved problems of bioinformatics. New high-accuracy methods of splicing sites recognition are needed to solve this problem. A question of current interest is to identify specific features of nucleotide sequences nearby splicing sites and recognize sites in sequence context. We performed a statistical analysis of human genes fragment database and revealed some characteristics of nucleotide sequences in splicing sites neighborhood. Frequencies of all nucleotides and dinucleotides in splicing sites environment were computed and nucleotides and dinucleotides with extremely high\\low occurrences were identified. Statistical information obtained in this work can be used in further development of the methods of splicing sites annotation and exon-intron structure recognition.

  11. Comparative Analysis of Splice Site Regions by Information Content

    Institute of Scientific and Technical Information of China (English)

    T. Shashi Rekha; Chanchal K. Mitra

    2006-01-01

    We have applied concepts from information theory for a comparative analysis of donor (gt) and acceptor (ag) splice site regions in the genes of five different organisms by calculating their mutual information content (relative entropy) over a selected block of nucleotides. A similar pattern that the information content decreases as the block size increases was observed for both regions in all the organisms studied. This result suggests that the information required for splicing might be contained in the consensus of ~6-8 nt at both regions. We assume from our study that even though the nucleotides are showing some degrees of conservation in the flanking regions of the splice sites, certain level of variability is still tolerated,which leads the splicing process to occur normally even if the extent of base pairing is not fully satisfied. We also suggest that this variability can be compensated by recognizing different splice sites with different spliceosomal factors.

  12. Splice Site Mutations in the ATP7A Gene

    DEFF Research Database (Denmark)

    Skjørringe, Tina; Tümer, Zeynep; Møller, Lisbeth Birk

    2011-01-01

    Menkes disease (MD) is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12...... mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation...... to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations...

  13. BAP1 missense mutation c.2054 A>T (p.E685V completely disrupts normal splicing through creation of a novel 5' splice site in a human mesothelioma cell line.

    Directory of Open Access Journals (Sweden)

    Arianne Morrison

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

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

    Directory of Open Access Journals (Sweden)

    Daniela Eckert

    2016-01-01

    Full Text Available The genome of the fission yeast Schizosaccharomyces pombe encodes 17 kinases that are essential for cell growth. These include the cell-cycle regulator Cdc2, as well as several kinases that coordinate cell growth, polarity, and morphogenesis during the cell cycle. In this study, we further characterized another of these essential kinases, Prp4, and showed that the splicing of many introns is dependent on Prp4 kinase activity. For detailed characterization, we chose the genes res1 and ppk8, each of which contains one intron of typical size and position. Splicing of the res1 intron was dependent on Prp4 kinase activity, whereas splicing of the ppk8 intron was not. Extensive mutational analyses of the 5' splice site of both genes revealed that proper transient interaction with the 5' end of snRNA U1 governs the dependence of splicing on Prp4 kinase activity. Proper transient interaction between the branch sequence and snRNA U2 was also important. Therefore, the Prp4 kinase is required for recognition and efficient splicing of introns displaying weak exon1/5' splice sites and weak branch sequences.

  15. Splice site mutations in the ATP7A gene.

    Directory of Open Access Journals (Sweden)

    Tina Skjørringe

    Full Text Available Menkes disease (MD is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12 mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations identified in 12 patients with milder phenotypes were predicted to have no significant effect on splicing, which concurs with the presence of wild-type transcript in 7 out of 9 patients investigated in vivo. Both the in silico predictions and the in vivo results support the hypothesis previously suggested by us and others, that the presence of some wild-type transcript is correlated to a milder phenotype.

  16. Sequence-specific flexibility organization of splicing flanking sequence and prediction of splice sites in the human genome.

    Science.gov (United States)

    Zuo, Yongchun; Zhang, Pengfei; Liu, Li; Li, Tao; Peng, Yong; Li, Guangpeng; Li, Qianzhong

    2014-09-01

    More and more reported results of nucleosome positioning and histone modifications showed that DNA structure play a well-established role in splicing. In this study, a set of DNA geometric flexibility parameters originated from molecular dynamics (MD) simulations were introduced to discuss the structure organization around splice sites at the DNA level. The obtained profiles of specific flexibility/stiffness around splice sites indicated that the DNA physical-geometry deformation could be used as an alternative way to describe the splicing junction region. In combination with structural flexibility as discriminatory parameter, we developed a hybrid computational model for predicting potential splicing sites. And the better prediction performance was achieved when the benchmark dataset evaluated. Our results showed that the mechanical deformability character of a splice junction is closely correlated with both the splice site strength and structural information in its flanking sequences.

  17. A new method for splice site prediction based on the sequence patterns of splicing signals and regulatory elements

    Institute of Scientific and Technical Information of China (English)

    SUN ZongXiao; SANG LingJie; JU LiNing; ZHU HuaiQiu

    2008-01-01

    It is of significance for splice site prediction to develop novel algorithms that combine the sequence patterns of regulatory elements such as enhancers and silencers with the patterns of splicing signals. In this paper, a statistical model of splicing signals was built based on the entropy density profile (EDP) method, weight array method (WAM) and κ test; moreover, the model of splicing regulatory elements was developed by an unsupervised self-learning method to detect motifs associated with regulatory elements. With two models incorporated, a multi-level support vector machine (SVM) system was de-vised to perform ab initio prediction for splice sites originating from DNA sequence in eukaryotic ge-home. Results of large scale tests on human genomic splice sites show that the new method achieves a comparative high performance in splice site prediction. The method is demonstrated to be with at least the same level of performance and usually better performance than the existing SpliceScan method based on modeling regulatory elements, and shown to have higher accuracies than the traditional methods with modeling splicing signals such as the GeneSplicer. In particular, the method has evident advantage over splice site prediction for the genes with lower GC content.

  18. Method of predicting Splice Sites based on signal interactions

    Directory of Open Access Journals (Sweden)

    Deogun Jitender S

    2006-04-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

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

    2015-12-15

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

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

    Science.gov (United States)

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

    2012-08-01

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

  1. Detection of Splice Sites Using Support Vector Machine

    Science.gov (United States)

    Varadwaj, Pritish; Purohit, Neetesh; Arora, Bhumika

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

  2. trans-splicing to spliceosomal U2 snRNA suggests disruption of branch site-U2 pairing during pre-mRNA splicing.

    Science.gov (United States)

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

    2007-06-22

    Pairing between U2 snRNA and the branch site of spliceosomal introns is essential for spliceosome assembly and is thought to be required for the first catalytic step of splicing. We have identified an RNA comprising the 5' end of U2 snRNA and the 3' exon of the ACT1-CUP1 reporter gene, resulting from a trans-splicing reaction in which a 5' splice site-like sequence in the universally conserved branch site-binding region of U2 is used in trans as a 5' splice site for both steps of splicing in vivo. Formation of this product occurs in functional spliceosomes assembled on reporter genes whose 5' splice sites are predicted to bind poorly at the spliceosome catalytic center. Multiple spatially disparate splice sites in U2 can be used, calling into question both the fate of its pairing to the branch site and the details of its role in splicing catalysis.

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

    DEFF Research Database (Denmark)

    Eden, E.; Brunak, Søren

    2004-01-01

    Prediction of splice sites in non-coding regions of genes is one of the most challenging aspects of gene structure recognition. We perform a rigorous analysis of such splice sites embedded in human 5' untranslated regions (UTRs), and investigate correlations between this class of splice sites...... and other features found in the adjacent exons and introns. By restricting the training of neural network algorithms to 'pure' UTRs (not extending partially into protein coding regions), we for the first time investigate the predictive power of the splicing signal proper, in contrast to conventional splice...... site prediction, which typically relies on the change in sequence at the transition from protein coding to non-coding. By doing so, the algorithms were able to pick up subtler splicing signals that were otherwise masked by 'coding' noise, thus enhancing significantly the prediction of 5' UTR splice...

  4. Splicing-site recognition of rice (Oryza sativa L.)DNA sequences by support vector machines

    Institute of Scientific and Technical Information of China (English)

    彭司华; 樊龙江; 彭小宁; 庄树林; 杜维; 陈良标

    2003-01-01

    Motivation: It was found that high accuracy splicing-site recognition of rice (Oryza sativa L.) DNA sequence is especially difficult. We described a new method for the splicing-site recognition of rice DNA sequences. Method: Based on the intron in eukaryotic organisms conforming to the principle of GT-AG, we used support vector machines (SVM) to predict the splicing sites. By machine learning, we built a model and used it to test the effect of the test data set of true and pseudo splicing sites. Results: The prediction accuracy we obtained was 87.53% at the true 5' end splicing site and 87.37% at the true 3' end splicing sites. The results suggested that the SVM approach could achieve higher accuracy than the previous approaches.

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

    Science.gov (United States)

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

    2015-01-01

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

  6. iSS-PseDNC: identifying splicing sites using pseudo dinucleotide composition.

    Science.gov (United States)

    Chen, Wei; Feng, Peng-Mian; Lin, Hao; Chou, Kuo-Chen

    2014-01-01

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

  7. The emergence of alternative 3' and 5' splice site exons from constitutive exons.

    Directory of Open Access Journals (Sweden)

    Eli Koren

    2007-05-01

    Full Text Available Alternative 3' and 5' splice site (ss events constitute a significant part of all alternative splicing events. These events were also found to be related to several aberrant splicing diseases. However, only few of the characteristics that distinguish these events from alternative cassette exons are known currently. In this study, we compared the characteristics of constitutive exons, alternative cassette exons, and alternative 3'ss and 5'ss exons. The results revealed that alternative 3'ss and 5'ss exons are an intermediate state between constitutive and alternative cassette exons, where the constitutive side resembles constitutive exons, and the alternative side resembles alternative cassette exons. The results also show that alternative 3'ss and 5'ss exons exhibit low levels of symmetry (frame-preserving, similar to constitutive exons, whereas the sequence between the two alternative splice sites shows high symmetry levels, similar to alternative cassette exons. In addition, flanking intronic conservation analysis revealed that exons whose alternative splice sites are at least nine nucleotides apart show a high conservation level, indicating intronic participation in the regulation of their splicing, whereas exons whose alternative splice sites are fewer than nine nucleotides apart show a low conservation level. Further examination of these exons, spanning seven vertebrate species, suggests an evolutionary model in which the alternative state is a derivative of an ancestral constitutive exon, where a mutation inside the exon or along the flanking intron resulted in the creation of a new splice site that competes with the original one, leading to alternative splice site selection. This model was validated experimentally on four exons, showing that they indeed originated from constitutive exons that acquired a new competing splice site during evolution.

  8. On splice site prediction using weight array models: a comparison of smoothing techniques

    Science.gov (United States)

    Taher, Leila; Meinicke, Peter; Morgenstern, Burkhard

    2007-11-01

    In most eukaryotic genes, protein-coding exons are separated by non-coding introns which are removed from the primary transcript by a process called "splicing". The positions where introns are cut and exons are spliced together are called "splice sites". Thus, computational prediction of splice sites is crucial for gene finding in eukaryotes. Weight array models are a powerful probabilistic approach to splice site detection. Parameters for these models are usually derived from m-tuple frequencies in trusted training data and subsequently smoothed to avoid zero probabilities. In this study we compare three different ways of parameter estimation for m-tuple frequencies, namely (a) non-smoothed probability estimation, (b) standard pseudo counts and (c) a Gaussian smoothing procedure that we recently developed.

  9. A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication

    Directory of Open Access Journals (Sweden)

    Stoltzfus C Martin

    2006-02-01

    Full Text Available Abstract Background Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1 primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss, which are positively regulated by exonic splicing enhancers (ESE and negatively regulated by exonic splicing silencers (ESS and intronic splicing silencers (ISS. We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production. Results Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3. Conclusion These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non

  10. Features of 5'-splice-site efficiency derived from disease-causing mutations and comparative genomics

    DEFF Research Database (Denmark)

    Roca, Xavier; Olson, Andrew J; Rao, Atmakuri R

    2007-01-01

    Many human diseases, including Fanconi anemia, hemophilia B, neurofibromatosis, and phenylketonuria, can be caused by 5'-splice-site (5'ss) mutations that are not predicted to disrupt splicing, according to position weight matrices. By using comparative genomics, we identify pairwise dependencies...

  11. Predicting mutually exclusive spliced exons based on exon length, splice site and reading frame conservation, and exon sequence homology

    Directory of Open Access Journals (Sweden)

    Hammesfahr Björn

    2011-06-01

    Full Text Available Abstract Background Alternative splicing of pre-mature RNA is an important process eukaryotes utilize to increase their repertoire of different protein products. Several types of different alternative splice forms exist including exon skipping, differential splicing of exons at their 3'- or 5'-end, intron retention, and mutually exclusive splicing. The latter term is used for clusters of internal exons that are spliced in a mutually exclusive manner. Results We have implemented an extension to the WebScipio software to search for mutually exclusive exons. Here, the search is based on the precondition that mutually exclusive exons encode regions of the same structural part of the protein product. This precondition provides restrictions to the search for candidate exons concerning their length, splice site conservation and reading frame preservation, and overall homology. Mutually exclusive exons that are not homologous and not of about the same length will not be found. Using the new algorithm, mutually exclusive exons in several example genes, a dynein heavy chain, a muscle myosin heavy chain, and Dscam were correctly identified. In addition, the algorithm was applied to the whole Drosophila melanogaster X chromosome and the results were compared to the Flybase annotation and an ab initio prediction. Clusters of mutually exclusive exons might be subsequent to each other and might encode dozens of exons. Conclusions This is the first implementation of an automatic search for mutually exclusive exons in eukaryotes. Exons are predicted and reconstructed in the same run providing the complete gene structure for the protein query of interest. WebScipio offers high quality gene structure figures with the clusters of mutually exclusive exons colour-coded, and several analysis tools for further manual inspection. The genome scale analysis of all genes of the Drosophila melanogaster X chromosome showed that WebScipio is able to find all but two of the 28

  12. The U2AF35-related protein Urp contacts the 3' splice site to promote U12-type intron splicing and the second step of U2-type intron splicing.

    Science.gov (United States)

    Shen, Haihong; Zheng, Xuexiu; Luecke, Stephan; Green, Michael R

    2010-11-01

    The U2AF35-related protein Urp has been implicated previously in splicing of the major class of U2-type introns. Here we show that Urp is also required for splicing of the minor class of U12-type introns. Urp is recruited in an ATP-dependent fashion to the U12-type intron 3' splice site, where it promotes formation of spliceosomal complexes. Remarkably, Urp also contacts the 3' splice site of a U2-type intron, but in this case is specifically required for the second step of splicing. Thus, through recognition of a common splicing element, Urp facilitates distinct steps of U2- and U12-type intron splicing.

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

    Science.gov (United States)

    Mattioli, Chiara; Pianigiani, Giulia; Pagani, Franco

    2013-10-01

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

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

    Science.gov (United States)

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

    2011-09-01

    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, Tra2β 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.

  15. Nonsense mutations and altered splice-site selection

    Energy Technology Data Exchange (ETDEWEB)

    Dietz, H.C. [Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States)

    1997-03-01

    The invited editorial by Maquat, regarding defects in RNA splicing and the consequence of shortened translational reading frames, provided a balanced and comprehensive review of the topic. We believe, however, that our work describing the nonsense codon-mediated skipping of fibrillin-1 exon 51 was interpreted in a manner that is not fully supported by our data. 6 refs.

  16. The strength of intron donor splice sites in human genes displays a bell-shaped pattern

    DEFF Research Database (Denmark)

    Wang, Kai; Wernersson, Rasmus; Brunak, Søren

    2011-01-01

    MOTIVATION: The gene concept has recently changed from the classical one protein notion into a much more diverse picture, where overlapping or fused transcripts, alternative transcription initiation, and genes within genes, add to the complexity generated by alternative splicing. Increased...... understanding of the mechanisms controlling pre-mRNA splicing is thus important for a wide range of aspects relating to gene expression. RESULTS: We have discovered a convex gene delineating pattern in the strength of 5' intron splice sites. When comparing the strengths of >18 000 intron containing Human genes......, we found that when analysing them separately according to the number of introns they contain, initial splice sites were always stronger on average than subsequent ones, and that a similar reversed trend exist towards the terminal gene part. The convex pattern is strongest for genes with up to 10...

  17. Sequence Analysis of In Vivo-Expressed HIV-1 Spliced RNAs Reveals the Usage of New and Unusual Splice Sites by Viruses of Different Subtypes

    Science.gov (United States)

    Vega, Yolanda; Delgado, Elena; de la Barrera, Jorge; Carrera, Cristina; Zaballos, Ángel; Cuesta, Isabel; Mariño, Ana; Ocampo, Antonio; Miralles, Celia; Pérez-Castro, Sonia; Álvarez, Hortensia; López-Miragaya, Isabel; García-Bodas, Elena; Díez-Fuertes, Francisco; Thomson, Michael M.

    2016-01-01

    HIV-1 RNAs are generated through a complex splicing mechanism, resulting in a great diversity of transcripts, which are classified in three major categories: unspliced, singly spliced (SS), and doubly spliced (DS). Knowledge on HIV-1 RNA splicing in vivo and by non-subtype B viruses is scarce. Here we analyze HIV-1 RNA splice site usage in CD4+CD25+ lymphocytes from HIV-1-infected individuals through pyrosequencing. HIV-1 DS and SS RNAs were amplified by RT-PCR in 19 and 12 samples, respectively. 13,108 sequences from HIV-1 spliced RNAs, derived from viruses of five subtypes (A, B, C, F, G), were identified. In four samples, three of non-B subtypes, five 3’ splice sites (3’ss) mapping to unreported positions in the HIV-1 genome were identified. Two, designated A4i and A4j, were used in 22% and 25% of rev RNAs in two viruses of subtypes B and A, respectively. Given their close proximity (one or two nucleotides) to A4c and A4d, respectively, they could be viewed as variants of these sites. Three 3’ss, designated A7g, A7h, and A7i, located 20, 32, and 18 nucleotides downstream of A7, respectively, were identified in a subtype C (A7g, A7h) and a subtype G (A7i) viruses, each in around 2% of nef RNAs. The new splice sites or variants of splice sites were associated with the usual sequence features of 3’ss. Usage of unusual 3’ss A4d, A4e, A5a, A7a, and A7b was also detected. A4f, previously identified in two subtype C viruses, was preferentially used by rev RNAs of a subtype C virus. These results highlight the great diversity of in vivo splice site usage by HIV-1 RNAs. The fact that four of five newly identified splice sites or variants of splice sites were detected in non-subtype B viruses allows anticipating an even greater diversity of HIV-1 splice site usage than currently known. PMID:27355361

  18. Sequence Analysis of In Vivo-Expressed HIV-1 Spliced RNAs Reveals the Usage of New and Unusual Splice Sites by Viruses of Different Subtypes.

    Directory of Open Access Journals (Sweden)

    Yolanda Vega

    Full Text Available HIV-1 RNAs are generated through a complex splicing mechanism, resulting in a great diversity of transcripts, which are classified in three major categories: unspliced, singly spliced (SS, and doubly spliced (DS. Knowledge on HIV-1 RNA splicing in vivo and by non-subtype B viruses is scarce. Here we analyze HIV-1 RNA splice site usage in CD4+CD25+ lymphocytes from HIV-1-infected individuals through pyrosequencing. HIV-1 DS and SS RNAs were amplified by RT-PCR in 19 and 12 samples, respectively. 13,108 sequences from HIV-1 spliced RNAs, derived from viruses of five subtypes (A, B, C, F, G, were identified. In four samples, three of non-B subtypes, five 3' splice sites (3'ss mapping to unreported positions in the HIV-1 genome were identified. Two, designated A4i and A4j, were used in 22% and 25% of rev RNAs in two viruses of subtypes B and A, respectively. Given their close proximity (one or two nucleotides to A4c and A4d, respectively, they could be viewed as variants of these sites. Three 3'ss, designated A7g, A7h, and A7i, located 20, 32, and 18 nucleotides downstream of A7, respectively, were identified in a subtype C (A7g, A7h and a subtype G (A7i viruses, each in around 2% of nef RNAs. The new splice sites or variants of splice sites were associated with the usual sequence features of 3'ss. Usage of unusual 3'ss A4d, A4e, A5a, A7a, and A7b was also detected. A4f, previously identified in two subtype C viruses, was preferentially used by rev RNAs of a subtype C virus. These results highlight the great diversity of in vivo splice site usage by HIV-1 RNAs. The fact that four of five newly identified splice sites or variants of splice sites were detected in non-subtype B viruses allows anticipating an even greater diversity of HIV-1 splice site usage than currently known.

  19. Overlapping cis sites used for splicing of HIV-1 env/nef and rev mRNAs.

    Science.gov (United States)

    Swanson, A K; Stoltzfus, C M

    1998-12-18

    Alternative splicing is used to generate more than 30 human immunodeficiency virus type 1 (HIV-1) spliced and unspliced mRNAs from a single primary transcript. The abundance of HIV-1 mRNAs is determined by the efficiencies with which its different 5' and 3' splice sites are used. Three splice sites (A4c, A4a, and A4b) are upstream of the rev initiator AUG. RNAs spliced at A4c, A4a, and A4b are used as mRNAs for Rev. Another 3' splice site (A5) is immediately downstream of the rev initiator. RNAs spliced at A5 are used as mRNAs for Env and Nef. In this report, primer extension analysis of splicing intermediates was used to show that there are eight branch points in this region, all of which map to adenosine residues. In addition, cis elements recognized by the cellular splicing machinery overlap; the two most 3' branch points overlap with the AG dinucleotides at rev 3' splice sites A4a and A4b. Competition of the overlapping cis sites for different splicing factors may play a role in maintaining the appropriate balance of mRNAs in HIV-1-infected cells. In support of this possibility, mutations at rev 3' splice site A4b AG dinucleotide dramatically increased splicing of the env/nef 3' splice site A5. This correlated with increased usage of the four most 3' branch points, which include those within the rev 3' splice site AG dinucleotides. Consistent with these results, analysis of a mutant in which three of the four env/nef branch points were inactivated indicated that use of splice site A5 was inhibited and splicing was shifted predominantly to the most 5' rev 3' splice site A4c with preferential use of the two most 5' branch points. Our results suggest that spliceosomes formed at rev A4a-4b, rev A4c, and env/nef A5 3' splice sites each recognize different subsets of the eight branch point sequences.

  20. Splicing-site recognition of rice (Oryza sativa L. ) DNA sequences by support vector machines

    Institute of Scientific and Technical Information of China (English)

    彭司华; 彭小宁; 庄树林; 杜维; 陈良标

    2003-01-01

    Motivation: It was found that high accuracy splicing-site recognition of rice ( Oryza satlva L. ) DNA sequence is especially difficult. We described a new method for the splicing-site recognition of rice DNA sequences. Method: Based on the intron in eukaryotic organisms conforming to the principle of GT-AG, we used support vector machines (SVM) to predict the splicing sites. By machine learning, we built a model and used it to test the effect of the test data set of true and pseudo splicing sites. Results : The prediction accuracy we obtained was 87.53% at the true 5' end splicing site and 87.37% at the true 3' end splicing sites. The results suggested that the SVM approach could achieve higher accuracy than the previous approaches.

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

    Science.gov (United States)

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

    2011-06-15

    In Caenorhabditis elegans, germ cells develop as spermatids in the larva and as oocytes in the adult. Such fundamentally different gametes are produced through a fine-tuned balance between feminizing and masculinizing genes. For example, the switch to oogenesis requires repression of the fem-3 mRNA through the mog genes. Here we report on the cloning and characterization of the sex determination gene mog-2. MOG-2 is the worm homolog of spliceosomal protein U2A'. We found that MOG-2 is expressed in most nuclei of somatic and germ cells. In addition to its role in sex determination, mog-2 is required for meiosis. Moreover, MOG-2 binds to U2B″/RNP-3 in the absence of RNA. We also show that MOG-2 associates with the U2 snRNA in the absence of RNP-3. Therefore, we propose that MOG-2 is a bona fide component of the U2 snRNP. Albeit not being required for general pre-mRNA splicing, MOG-2 increases the splicing efficiency to a cryptic splice site that is located at the 5' end of the exon. Copyright © 2011. Published by Elsevier Inc.

  2. Nucleotide sequence composition adjacent to intronic splice sites improves splicing efficiency via its effect on pre-mRNA local folding in fungi.

    Science.gov (United States)

    Zafrir, Zohar; Tuller, Tamir

    2015-10-01

    RNA splicing is the central process of intron removal in eukaryotes known to regulate various cellular functions such as growth, development, and response to external signals. The canonical sequences indicating the splicing sites needed for intronic boundary recognition are well known. However, the roles and evolution of the local folding of intronic and exonic sequence features adjacent to splice sites has yet to be thoroughly studied. Here, focusing on four fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, and Candida albicans), we performed for the first time a comprehensive high-resolution study aimed at characterizing the encoding of intronic splicing efficiency in pre-mRNA transcripts and its effect on intron evolution. Our analysis supports the conjecture that pre-mRNA local folding strength at intronic boundaries is under selective pressure, as it significantly affects splicing efficiency. Specifically, we show that in the immediate region of 12-30 nucleotides (nt) surrounding the intronic donor site there is a preference for weak pre-mRNA folding; similarly, in the region of 15-33 nt surrounding the acceptor and branch sites there is a preference for weak pre-mRNA folding. We also show that in most cases there is a preference for strong pre-mRNA folding further away from intronic splice sites. In addition, we demonstrate that these signals are not associated with gene-specific functions, and they correlate with splicing efficiency measurements (r = 0.77, P = 2.98 × 10(-21)) and with expression levels of the corresponding genes (P = 1.24 × 10(-19)). We suggest that pre-mRNA folding strength in the above-mentioned regions has a direct effect on splicing efficiency by improving the recognition of intronic boundaries. These new discoveries are contributory steps toward a broader understanding of splicing regulation and intronic/transcript evolution.

  3. Identification and characterization of NF1 splicing mutations in Korean patients with neurofibromatosis type 1.

    Science.gov (United States)

    Jang, Mi-Ae; Kim, Young-Eun; Kim, Sun Kyung; Lee, Myoung-Keun; Kim, Jong-Won; Ki, Chang-Seok

    2016-08-01

    Neurofibromatosis type I (NF1) is an autosomal dominant genetic disorder caused by NF1 mutations. Although mutations affecting mRNA splicing are the most common molecular defects in NF1, few studies have analyzed genomic DNA (gDNA)-mRNA correlations in Korean NF1 patients. In this study, we investigated 28 unrelated NF1 patients who showed splicing alterations in reverse transcription-PCR of NF1 mRNA and identified 24 different NF1 splicing mutations, 9 of which were novel. These mutations can be categorized into five groups: exon skipping resulting from mutations at authentic 5' and 3' splice sites (type I, 46%), cryptic exon inclusion caused by deep intronic mutations (type II, 8%), creation of new splice sites causing loss of exonic sequences (type III, 8%), activation of cryptic splice sites due to disruption of authentic splice sites (type IV, 25%) and exonic sequence alterations causing exon skipping (type V, 13%). In total, 42% of all splicing mutations did not involve the conserved AG/GT dinucleotides of the splice sites, making it difficult to identify the correct mutation sites at the gDNA level. These results add to the mutational spectrum of NF1 and further elucidate the gDNA-mRNA correlations of NF1 mutations.

  4. Suppression of 5' splice-sites through multiple exonic motifs by hnRNP L.

    Science.gov (United States)

    Loh, Tiing Jen; Choi, Namjeong; Moon, Heegyum; Jang, Ha Na; Liu, Yongchao; Zhou, Jianhua; Zheng, Xuexiu; Shen, Haihong

    2017-03-01

    Selection of 5' splice-sites (5'SS) in alternative splicing plays an important role in gene regulation. Although regulatory mechanisms of heterogeneous nuclear ribonucleoprotein L (hnRNP L), a well-known splicing regulatory protein, have been studied in a substantial level, its role in 5'SS selection is not thoroughly defined. By using a KLF6 pre-mRNA alternative splicing model, we demonstrate in this report that hnRNP L inhibits proximal 5'SS but promotes two consecutive distal 5'SS splicing, antagonizing SRSF1 roles in KLF6 pre-mRNA splicing. In addition, three consecutive CA-rich sequences in a CA cassette immediately upstream of the proximal 5'SS are all required for hnRNP L functions. Importantly, the CA-cassette locations on the proximal exon do not affect hnRNP L roles. We further show that the proximal 5'SS but not the two distal 5'SSs are essential for hnRNP L activities. Notably, in a Bcl-x pre-mRNA model that contains two alternative 5'SS but includes CA-rich elements at distal exon, we demonstrate that hnRNP L also suppresses nearby 5'SS activation. Taken together, we conclude that hnRNP L suppresses 5'SS selection through multiple exonic motifs.

  5. Site-specific reverse splicing of a HEG-containing group I intron in ribosomal RNA

    Science.gov (United States)

    Birgisdottir, Åsa B.; Johansen, Steinar

    2005-01-01

    The wide, but scattered distribution of group I introns in nature is a result of two processes; the vertical inheritance of introns with or without losses, and the occasional transfer of introns across species barriers. Reversal of the group I intron self-splicing reaction, termed reverse splicing, coupled with reverse transcription and genomic integration potentially mediate an RNA-based intron mobility pathway. Compared to the well characterized endonuclease-mediated intron homing, reverse splicing is less specific and represents a likely explanation for many intron transpositions into new genomic sites. However, the frequency and general role of an RNA-based mobility pathway in the spread of natural group I introns is still unclear. We have used the twin-ribozyme intron (Dir.S956-1) from the myxomycete Didymium iridis to test how a mobile group I intron containing a homing endonuclease gene (HEG) selects between potential insertion sites in the small subunit (SSU) rRNA in vitro, in Escherichia coli and in yeast. Surprisingly, the results show a site-specific RNA-based targeting of Dir.S956-1 into its natural (S956) SSU rRNA site. Our results suggest that reverse splicing, in addition to the established endonuclease-mediated homing mechanism, potentially accounts for group I intron spread into the homologous sites of different strains and species. PMID:15817568

  6. Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1

    Directory of Open Access Journals (Sweden)

    Tobias Bonifert

    2016-01-01

    Full Text Available Inherited optic neuropathies (ION present an important cause of blindness in the European working-age population. Recently we reported the discovery of four independent families with deep intronic mutations in the main inherited optic neuropathies gene OPA1. These deep intronic mutations cause mis-splicing of the OPA1 pre-messenger-RNA transcripts by creating cryptic acceptor splice sites. As a rescue strategy we sought to prevent mis-splicing of the mutant pre-messenger-RNA by applying 2′O-methyl-antisense oligonucleotides (AONs with a full-length phosphorothioate backbone that target the cryptic acceptor splice sites and the predicted novel branch point created by the deep intronic mutations, respectively. Transfection of patient-derived primary fibroblasts with these AONs induced correct splicing of the mutant pre-messenger-RNA in a time and concentration dependent mode of action, as detected by pyrosequencing of informative heterozygous variants. The treatment showed strong rescue effects (≃55% using the cryptic acceptor splice sites targeting AON and moderate rescue (≃16% using the branch point targeting AON. The highest efficacy of Splice correction could be observed 4 days after treatment however, significant effects were still seen 14 days post-transfection. Western blot analysis revealed increased amounts of OPA1 protein with maximum amounts at ≃3 days post-treatment. In summary, we provide the first mutation-specific in vitro rescue strategy for OPA1 deficiency using synthetic AONs.

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

    DEFF Research Database (Denmark)

    Wang, Kai; Ussery, David; Brunak, Søren

    2009-01-01

    , splice site prediction program called NetAspGene, for the genus Aspergillus. Gene sequences from Aspergillus fumigatus, the most common mould pathogen, were used to build and test our model. Compared to many animals and plants, Aspergillus contains smaller introns; thus we have applied a larger window...

  8. Transposable elements in disease-associated cryptic exons.

    Science.gov (United States)

    Vorechovsky, Igor

    2010-02-01

    Transposable elements (TEs) make up a half of the human genome, but the extent of their contribution to cryptic exon activation that results in genetic disease is unknown. Here, a comprehensive survey of 78 mutation-induced cryptic exons previously identified in 51 disease genes revealed the presence of TEs in 40 cases (51%). Most TE-containing exons were derived from short interspersed nuclear elements (SINEs), with Alus and mammalian interspersed repeats (MIRs) covering >18 and >16% of the exonized sequences, respectively. The majority of SINE-derived cryptic exons had splice sites at the same positions of the Alu/MIR consensus as existing SINE exons and their inclusion in the mRNA was facilitated by phylogenetically conserved changes that improved both traditional and auxiliary splicing signals, thus marking intronic TEs amenable for pathogenic exonization. The overrepresentation of MIRs among TE exons is likely to result from their high average exon inclusion levels, which reflect their strong splice sites, a lack of splicing silencers and a high density of enhancers, particularly (G)AA(G) motifs. These elements were markedly depleted in antisense Alu exons, had the most prominent position on the exon-intron gradient scale and are proposed to promote exon definition through enhanced tertiary RNA interactions involving unpaired (di)adenosines. The identification of common mechanisms by which the most dynamic parts of the genome contribute both to new exon creation and genetic disease will facilitate detection of intronic mutations and the development of computational tools that predict TE hot-spots of cryptic exon activation.

  9. G runs in cystathionine beta-synthase c.833C/c.844_845ins68 mRNA are splicing silencers of pathogenic 3' splice sites.

    Science.gov (United States)

    Romano, Maurizio

    2010-08-01

    The c.844_845ins68 is an evolutionary conserved polymorphism of the cystathionine beta-synthase gene that segregates with the pathogenic c.833C mutation and consists of a 68nt insertion duplicating the 3' splice site between intron 7 and exon 8. The gene rearrangement brought two GGGG runs close to each other and generated a splicing control element that allows the constitutive selection of the more distal 3' splice site in the c.844_854ins68 carriers. In this study, we have characterized functionally the two G4 runs within the duplication and have found that they work as silencers of the upstream potentially pathogenic 3' splice sites has been functionally characterized. This selection allows skipping of both the 68nt-insertion and the c.833C mutation, and is essential to preserve the wild-type ORF. Knocking down hnRNP H and F expression modulated the rescue of the proximal 3' splice site more than hnRNP H alone. These observations suggest that hnRNP H/F contribute jointly to prevention of CBS deficiency in c.844_854ins68 carriers by silencing the potentially pathogenic upstream acceptor site. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

    Directory of Open Access Journals (Sweden)

    Jonàs Juan-Mateu

    Full Text Available DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements.

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

    Science.gov (United States)

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

    2005-06-01

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

  12. Original tandem duplication in FXIIIA gene with splicing site modification and four amino acids insertion causes factor XIII deficiency.

    Science.gov (United States)

    Louhichi, Nacim; Haj Salem, Ikhlass; Medhaffar, Moez; Miled, Nabil; Hadji, Ahmad F; Keskes, Leila; Fakhfakh, Faiza

    2017-04-01

    : Recessive mutations of F13A gene are reported to be responsible of FXIIIA subunit deficiency (FXIIIA). In all, some intronic nucleotide changes identified in this gene were investigated by in-silico analysis and occasionally supported by experimental data or reported in some cases as a polymorphism. To determine the molecular defects responsible of congenital factor XIII deficiency in Libyan patient, molecular analysis was performed by direct DNA sequencing of the coding regions and splice junctions of the FXIIIA subunit gene (F13A). A splicing minigene assay was used to study the effect of this mutation. Bioinformatics exploration was fulfilled to conceive consequences on protein. A 12-bp duplication straddling the border of intron 9 and exon 10 leads to two 3' acceptor splice sites, resulting in silencing of the downstream wild 3' splice site. It caused an in-frame insertion of 12 nucleotides into mRNA and four amino acids into protein. Bioinformatic analysis predicts that the insertion of four amino acids affects the site 3 of calcium binding site, which disturbs the smooth function of the FXIIIA peptide causing the factor XIII deficiency. This study showed that a small duplication seems to weaken the original 3' splice site and enhance the activation of a new splice site responsible for an alternative splicing. It would be interesting to examine the underlying molecular mechanism involved in this rearrangement.

  13. Quantitation of normal CFTR mRNA in CF patients with splice-site mutations

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Z.; Olsen, J.C.; Silverman, L.M. [Univ. of North Carolina, Chapel Hill, NC (United States)] [and others

    1994-09-01

    Previously we identified two mutations in introns of the CFTR gene associated with partially active splice sites and unusual clinical phenotypes. One mutation in intron 19 (3849+10 kb C to T) is common in CF patients with normal sweat chloride values; an 84 bp sequence from intron 19, which contains a stop codon, is inserted between exon 19 and exon 20 in most nasal CFTR transcripts. The other mutation in intron 14B (2789+5 G to A) is associated with elevated sweat chloride levels, but mild pulmonary disease; exon 14B (38 bp) is spliced out of most nasal CFTR transcipts. The remaining CFTR cDNA sequences, other than the 84 bp insertion of exon 14B deletion, are identical to the published sequence. To correlate genotype and phenotype, we used quantitative RT-PCR to determine the levels of normally-spliced CFTR mRNA in nasal epithelia from these patients. CFTR cDNA was amplified (25 cycles) by using primers specific for normally-spliced species, {gamma}-actin cDNA was amplified as a standard.

  14. Novel splice site mutation in keratin 1 underlies mild epidermolytic palmoplantar keratoderma in three kindreds.

    Science.gov (United States)

    Hatsell, S J; Eady, R A; Wennerstrand, L; Dopping-Hepenstal, P; Leigh, I M; Munro, C; Kelsell, D P

    2001-04-01

    We report a novel mutation in the exon 6 splice donor site of keratin 1 (G4134A) that segregates with a palmoplantar keratoderma in three kindreds. The nucleotide substitution leads to the utilization of a novel in-frame splice site 54 bases downstream of the mutation with the subsequent insertion of 18 amino acids into the 2B rod domain. This mutation appears to have a milder effect than previously described mutations in the helix initiation and termination sequence on the function of the rod domain, with regard to filament assembly and stability. Affected individuals displayed only mild focal epidermolysis in the spinous layer of palmoplantar epidermis, in comparison with cases of bullous congenital ichthyosiform erythroderma also due to keratin 1 mutations, which show widespread and severe epidermolysis. This study describes a novel mutation in KRT1 that results in a phenotype distinct from classical bullous congenital ichthyosiform erythroderma.

  15. Splice site mutations in mismatch repair genes and risk of cancer in the general population

    DEFF Research Database (Denmark)

    Thomsen, Mette; Nordestgaard, Børge G; Tybjærg-Hansen, Anne;

    2013-01-01

    We tested the hypothesis that splice site variations in MSH2 and MLH1 are associated with increased risk of hereditary non-polyposis colorectal cancer (HNPCC) and of cancer in general in the general population. In a cohort of 154 HNPCC patients with sequenced MSH2 and MLH1, we identified four...... possible splice-site mutations, which we subsequently genotyped in more than 9,000 individuals from the general population. Allele frequencies in the general population were 0 % for 942+3A>T in MSH2, 0.05 % for 307-19A>G, 0.005 % for 1,667+(2-8)del(taaatca);ins(attt), and 4.4 % for 1039-8T>A in MLH1. Odds...

  16. Global identification of hnRNP A1 binding sites for SSO-based splicing modulation

    DEFF Research Database (Denmark)

    Bruun, Gitte H; Doktor, Thomas K; Borch-Jensen, Jonas;

    2016-01-01

    for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. RESULTS: Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing...... regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP......-identified hnRNP A1 binding site immediately downstream of the 5' splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site...

  17. 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

    Energy Technology Data Exchange (ETDEWEB)

    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

    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

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

    Science.gov (United States)

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  20. Splicing mosaic of the myophosphorylase gene due to a silent mutation in McArdle disease.

    Science.gov (United States)

    Fernandez-Cadenas, I; Andreu, A L; Gamez, J; Gonzalo, R; Martín, M A; Rubio, J C; Arenas, J

    2003-11-25

    The authors report the molecular findings in a patient with McArdle disease who harbored a silent polymorphism (K608K) in the myophosphorylase gene. cDNA studies demonstrated that this polymorphism leads to a severe mosaic alteration in mRNA splicing, including exon skipping, activation of cryptic splice-sites, and exon-intron reorganizations. These findings suggest that, in patients with McArdle disease in whom no pathogenic mutation has been found, any a priori silent polymorphism should be re-evaluated as a putative splicing mutation.

  1. Interpretation of mRNA splicing mutations in genetic disease: review of the literature and guidelines for information-theoretical analysis [v2; ref status: indexed, http://f1000r.es/54y

    Directory of Open Access Journals (Sweden)

    Natasha G. Caminsky

    2015-03-01

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

  2. Interpretation of mRNA splicing mutations in genetic disease: review of the literature and guidelines for information-theoretical analysis [v1; ref status: indexed, http://f1000r.es/4nq

    Directory of Open Access Journals (Sweden)

    Natasha Caminsky

    2014-11-01

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

  3. Novel Infection Site and Ecology of Cryptic Didymocystis sp. (Trematoda) in the Fish Scomberomorus maculatus.

    Science.gov (United States)

    Schrandt, Meagan N; Andres, Michael J; Powers, Sean P; Overstreet, Robin M

    2016-06-01

    An undescribed, cryptic species of Didymocystis, as determined from sequences of 2 ribosomal genes and superficially similar to Didymocystis scomberomori ( MacCallum and MacCallum, 1916 ), infected the skin of the Spanish mackerel, Scomberomorus maculatus , in the north-central Gulf of Mexico (GOM). An analysis of 558 fish from 2011 to 2013 from Louisiana, Mississippi, Alabama, and the Florida panhandle showed the prevalence of the trematode varied both spatially and temporally but not with sex of the fish host. Month, year, and geographic location were identified by a negative binomial generalized linear model as indicators of the abundance and intensity of infection. Prevalence, abundance, and intensity of infection were greatest in spring and fall months off the Florida panhandle. Furthermore, the abundance and intensity of infection correlated negatively with fork length, weight, and gonad weight of mature fish but positively with longitude. Therefore, smaller adult fish tended to be more infected than larger adults, and prevalence and intensity increased from west to east (Louisiana to Florida). Spatial and temporal trends seemed to result from physical factors (e.g., water temperature, salinity, bottom type), but they also coincided with the annual migration of S. maculatus as fish moved northward along the GOM coastline from the southern tip of Florida in the spring months and returned in the fall, being present in the north-central GOM from late spring through fall. This pattern suggests the possibility that acquisition of infections occurred from a molluscan host in waters off the Florida panhandle.

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

    NARCIS (Netherlands)

    Garanto, A.; Chung, D.C.; Duijkers, L.; Corral-Serrano, J.C.; Messchaert, M.; Xiao, R.; Bennett, J.; Vandenberghe, L.H.; Collin, R.W.J.

    2016-01-01

    Leber congenital amaurosis (LCA) is a severe disorder resulting in visual impairment usually starting in the first year of life. The most frequent genetic cause of LCA is an intronic mutation in CEP290 (c.2991 + 1655A > G) that creates a cryptic splice donor site resulting in the insertion of a p

  5. Differential splicing of human androgen receptor pre-mRNA in X-linked reifenstein syndrome, because of a deletion involving a putative branch site

    Energy Technology Data Exchange (ETDEWEB)

    Ris-Stalpers, C.; Verleun-Mooijman, M.C.T.; Blaeij, T.J.P. de; Brinkmann, A.O.; Degenhart, H.J.; Trapman, J. (Erasmus Univ., Rotterdam (Netherlands))

    1994-04-01

    The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of >6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-point sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS. 42 refs., 6 figs., 1 tab.

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

    Science.gov (United States)

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

    1996-04-01

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

  7. iSS-PC: Identifying Splicing Sites via Physical-Chemical Properties Using Deep Sparse Auto-Encoder.

    Science.gov (United States)

    Xu, Zhao-Chun; Wang, Peng; Qiu, Wang-Ren; Xiao, Xuan

    2017-08-15

    Gene splicing is one of the most significant biological processes in eukaryotic gene expression, such as RNA splicing, which can cause a pre-mRNA to produce one or more mature messenger RNAs containing the coded information with multiple biological functions. Thus, identifying splicing sites in DNA/RNA sequences is significant for both the bio-medical research and the discovery of new drugs. However, it is expensive and time consuming based only on experimental technique, so new computational methods are needed. To identify the splice donor sites and splice acceptor sites accurately and quickly, a deep sparse auto-encoder model with two hidden layers, called iSS-PC, was constructed based on minimum error law, in which we incorporated twelve physical-chemical properties of the dinucleotides within DNA into PseDNC to formulate given sequence samples via a battery of cross-covariance and auto-covariance transformations. In this paper, five-fold cross-validation test results based on the same benchmark data-sets indicated that the new predictor remarkably outperformed the existing prediction methods in this field. Furthermore, it is expected that many other related problems can be also studied by this approach. To implement classification accurately and quickly, an easy-to-use web-server for identifying slicing sites has been established for free access at: http://www.jci-bioinfo.cn/iSS-PC.

  8. Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

    Directory of Open Access Journals (Sweden)

    Kim Dong Seon

    2012-11-01

    Full Text Available Abstract Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution.

  9. Cryptic Protein Priming Sites in Two Different Domains of Duck Hepatitis B Virus Reverse Transcriptase for Initiating DNA Synthesis In Vitro▿

    Science.gov (United States)

    Boregowda, Rajeev K.; Lin, Li; Zhu, Qin; Tian, Fang; Hu, Jianming

    2011-01-01

    Initiation of reverse transcription in hepadnaviruses is accomplished by a unique protein-priming mechanism whereby a specific Y residue in the terminal protein (TP) domain of the viral reverse transcriptase (RT) acts as a primer to initiate DNA synthesis, which is carried out by the RT domain of the same protein. When separate TP and RT domains from the duck hepatitis B virus (DHBV) RT protein were tested in a trans-complementation assay in vitro, the RT domain could also serve, unexpectedly, as a protein primer for DNA synthesis, as could a TP mutant lacking the authentic primer Y (Y96) residue. Priming at these other, so-called cryptic, priming sites in both the RT and TP domains shared the same requirements as those at Y96. A mini RT protein with both the TP and RT domains linked in cis, as well as the full-length RT protein, could also initiate DNA synthesis using cryptic priming sites. The cryptic priming site(s) in TP was found to be S/T, while those in the RT domain were Y and S/T. As with the authentic TP Y96 priming site, the cryptic priming sites in the TP and RT domains could support DNA polymerization subsequent to the initial covalent linkage of the first nucleotide to the priming amino acid residue. These results provide new insights into the complex mechanisms of protein priming in hepadnaviruses, including the selection of the primer residue and the interactions between the TP and RT domains that is essential for protein priming. PMID:21593164

  10. Tissue-specific transcription start sites and alternative splicing of the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor gene: a new PTH/PTHrP receptor splice variant that lacks the signal peptide.

    Science.gov (United States)

    Joun, H; Lanske, B; Karperien, M; Qian, F; Defize, L; Abou-Samra, A

    1997-04-01

    The PTH/PTHrP receptor gene is expressed in bone and kidney as well as in many other tissues. Using primer extension followed by rapid cloning of amplified complementary DNA ends, we have isolated new PTH/PTHrP receptor complementary DNAs with different splicing patterns and have characterized a new upstream transcription start site. Three 5' nontranslated exons, U3, U2 and U1, located 4.8, 2.5, and 1.2 kb upstream of the exon that encodes the putative signal peptide of the classical receptor (exon S), have been characterized. Four types of splicing patterns were recognized. Type I splicing pattern is transcribed from exon U1 and is spliced to exons S and E1; this pattern was found in most tissues tested. Types II, III, and IV splicing patterns are transcribed from exon U3 and have a restricted tissue distribution. Type II splice pattern, containing exons U3, U2, and S and type III splicing pattern, containing exon U3, U2, and E1 (skipping exon S), was found only in kidney. Type IV splice pattern, containing exon U3 and S was found both in kidney and ovary. Because the type III splice variant skips exon S, translation of this splice variant initiates at a different AUG codon. The type III splice variant was weakly expressed on the cell surface of COS-7 cells, as assessed by double antibody binding assay, and no detectable ligand binding was observed on intact cells. The type III splice variant, however, increased cAMP accumulation in COS-7 cells when challenged with PTH(1-34), PTH(1-84) and hPTHrP(1-36) with EC50s that are similar to those observed in COS-7 cells expressing the type I variant but with a maximum stimulation that was lower than that observed in COS-7 cells expressing the type I variant. These data indicate low levels of cell surface expression of the type III splice variant. Treatment of COS-7 cells with tunicamycin decreased the size of the type I splice variant from a broad band of 85 kDa to a compact band of about 60 kDa. The type III splice

  11. A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo

    DEFF Research Database (Denmark)

    Damgaard, Christian Kroun; Kahns, Søren; Lykke-Andersen, Søren;

    2008-01-01

    Transcription and pre-mRNA splicing are interdependent events. Although mechanisms governing the effects of transcription on splicing are becoming increasingly clear, the means by which splicing affects transcription remain elusive. Using cell lines stably expressing HIV-1 or β-globin mRNAs, harb...

  12. K2P2.1 (TREK-1)–activator complexes reveal a cryptic selectivity filter binding site

    Energy Technology Data Exchange (ETDEWEB)

    Lolicato, Marco; Arrigoni, Cristina; Mori, Takahiro; Sekioka, Yoko; Bryant, Clifford; Clark, Kimberly A.; Minor, Jr , Daniel L. (Ono); (UCSF)

    2017-07-10

    Polymodal thermo- and mechanosensitive two-pore domain potassium (K2P) channels of the TREK1 subfamily generate ‘leak’ currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perception and anaesthetic responses1, 2, 3. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore-forming regions per subunit4, 5, 6. In contrast to other potassium channels, K2P channels use a selectivity filter ‘C-type’ gate7, 8, 9, 10 as the principal gating site. Despite recent advances3, 11, 12, poor pharmacological profiles of K2P channels limit mechanistic and biological studies. Here we describe a class of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1 (also known as TREK-1) alone and with two selective K2P2.1 (TREK-1) and K2P10.1 (TREK-2) activators—an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402—define a cryptic binding pocket unlike other ion channel small-molecule binding sites and, together with functional studies, identify a cation–π interaction that controls selectivity. Together, our data reveal a druggable K2P site that stabilizes the C-type gate ‘leak mode’ and provide direct evidence for K2P selectivity filter gating.

  13. Regulation of Vif mRNA splicing by human immunodeficiency virus type 1 requires 5' splice site D2 and an exonic splicing enhancer to counteract cellular restriction factor APOBEC3G.

    Science.gov (United States)

    Mandal, Dibyakanti; Exline, Colin M; Feng, Zehua; Stoltzfus, C Martin

    2009-06-01

    The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5' splice site (5'ss) D1, to the first splice acceptor, 3'ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5'ss D2, which is 50 nucleotides downstream of 3'ss A1; a GGGG silencer motif proximal to 5'ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5'ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3'ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5'ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5'ss D2 and ESEVif are required for production of

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

    Science.gov (United States)

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

    2012-01-01

    Background The aim of this study was to characterize the genetic etiology in a patient who presented with permanent neonatal diabetes at 2 months of age. Methodology/Principal Findings Regulatory elements and coding exons 2 and 3 of the INS gene were amplified and sequenced from genomic and complementary DNA samples. A novel heterozygous INS mutation within the terminal intron of the gene was identified in the proband and her affected father. This mutation introduces an ectopic splice site leading to the insertion of 29 nucleotides from the intronic sequence into the mature mRNA, which results in a longer and abnormal transcript. Conclusions/Significance This study highlights the importance of routinely sequencing the exon-intron boundaries and the need to carry out additional studies to confirm the pathogenicity of any identified intronic genetic variants. PMID:22235272

  15. Permanent neonatal diabetes caused by creation of an ectopic splice site within the INS gene.

    Directory of Open Access Journals (Sweden)

    Intza Garin

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

  16. Partial deficiency of emerin caused by a splice site mutation in EMD.

    Science.gov (United States)

    Yuan, Junhui; Ando, Masahiro; Higuchi, Itsuro; Sakiyama, Yusuke; Matsuura, Eiji; Michizono, Kumiko; Watanabe, Osamu; Nagano, Shinjiro; Inamori, Yukie; Hashiguchi, Akihiro; Higuchi, Yujiro; Yoshimura, Akiko; Takashima, Hiroshi

    2014-01-01

    Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the EMD gene on the X chromosome, which codes for emerin, an inner nuclear membrane protein. Monoclonal antibodies against the N-terminus of emerin protein are used to screen for emerin deficiency in clinical practice. However, these tests may not accurately reflect the disease in some cases. We herein describe the identification of a splice site mutation in the EMD gene in a Japanese patient who suffered from complete atrioventricular conduction block, mild muscle weakness and joint contracture, and a persistently elevated serum creatine kinase level. We used multiple antibodies to confirm the presence of a novel truncating mutation in emerin without the transmembrane region and C-terminus in the skeletal muscle.

  17. Prediction of primate splice site using inhomogeneous Markov chain and neural network.

    Science.gov (United States)

    Liu, Libin; Ho, Yee-Kin; Yau, Stephen

    2007-07-01

    The inhomogeneous Markov chain model is used to discriminate acceptor and donor sites in genomic DNA sequences. It outperforms statistical methods such as homogeneous Markov chain model, higher order Markov chain and interpolated Markov chain models, and machine-learning methods such as k-nearest neighbor and support vector machine as well. Besides its high accuracy, another advantage of inhomogeneous Markov chain model is its simplicity in computation. In the three states system (acceptor, donor, and neither), the inhomogeneous Markov chain model is combined with a three-layer feed forward neural network. Using this combined system 3175 primate splice-junction gene sequences have been tested, with a prediction accuracy of greater than 98%.

  18. Identification of new splice sites used for generation of rev transcripts in human immunodeficiency virus type 1 subtype C primary isolates.

    Directory of Open Access Journals (Sweden)

    Elena Delgado

    Full Text Available The HIV-1 primary transcript undergoes a complex splicing process by which more than 40 different spliced RNAs are generated. One of the factors contributing to HIV-1 splicing complexity is the multiplicity of 3' splice sites (3'ss used for generation of rev RNAs, with two 3'ss, A4a and A4b, being most commonly used, a third site, A4c, used less frequently, and two additional sites, A4d and A4e, reported in only two and one isolates, respectively. HIV-1 splicing has been analyzed mostly in subtype B isolates, and data on other group M clades are lacking. Here we examine splice site usage in three primary isolates of subtype C, the most prevalent clade in the HIV-1 pandemic, by using an in vitro infection assay of peripheral blood mononuclear cells. Viral spliced RNAs were identified by RT-PCR amplification using a fluorescently-labeled primer and software analyses and by cloning and sequencing the amplified products. The results revealed that splice site usage for generation of rev transcripts in subtype C differs from that reported for subtype B, with most rev RNAs using two previously unreported 3'ss, one located 7 nucleotides upstream of 3'ss A4a, designated A4f, preferentially used by two isolates, and another located 14 nucleotides upstream of 3'ss A4c, designated A4g, preferentially used by the third isolate. A new 5' splice site, designated D2a, was also identified in one virus. Usage of the newly identified splice sites is consistent with sequence features commonly found in subtype C viruses. These results show that splice site usage may differ between HIV-1 subtypes.

  19. Identification of new splice sites used for generation of rev transcripts in human immunodeficiency virus type 1 subtype C primary isolates.

    Science.gov (United States)

    Delgado, Elena; Carrera, Cristina; Nebreda, Paloma; Fernández-García, Aurora; Pinilla, Milagros; García, Valentina; Pérez-Álvarez, Lucía; Thomson, Michael M

    2012-01-01

    The HIV-1 primary transcript undergoes a complex splicing process by which more than 40 different spliced RNAs are generated. One of the factors contributing to HIV-1 splicing complexity is the multiplicity of 3' splice sites (3'ss) used for generation of rev RNAs, with two 3'ss, A4a and A4b, being most commonly used, a third site, A4c, used less frequently, and two additional sites, A4d and A4e, reported in only two and one isolates, respectively. HIV-1 splicing has been analyzed mostly in subtype B isolates, and data on other group M clades are lacking. Here we examine splice site usage in three primary isolates of subtype C, the most prevalent clade in the HIV-1 pandemic, by using an in vitro infection assay of peripheral blood mononuclear cells. Viral spliced RNAs were identified by RT-PCR amplification using a fluorescently-labeled primer and software analyses and by cloning and sequencing the amplified products. The results revealed that splice site usage for generation of rev transcripts in subtype C differs from that reported for subtype B, with most rev RNAs using two previously unreported 3'ss, one located 7 nucleotides upstream of 3'ss A4a, designated A4f, preferentially used by two isolates, and another located 14 nucleotides upstream of 3'ss A4c, designated A4g, preferentially used by the third isolate. A new 5' splice site, designated D2a, was also identified in one virus. Usage of the newly identified splice sites is consistent with sequence features commonly found in subtype C viruses. These results show that splice site usage may differ between HIV-1 subtypes.

  20. 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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-09-01

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

  1. The CUGBP2 splicing factor regulates an ensemble of branchpoints from perimeter binding sites with implications for autoregulation.

    Directory of Open Access Journals (Sweden)

    Jill A Dembowski

    2009-08-01

    Full Text Available Alternative pre-mRNA splicing adjusts the transcriptional output of the genome by generating related mRNAs from a single primary transcript, thereby expanding protein diversity. A fundamental unanswered question is how splicing factors achieve specificity in the selection of target substrates despite the recognition of information-poor sequence motifs. The CUGBP2 splicing regulator plays a key role in the brain region-specific silencing of the NI exon of the NMDA R1 receptor. However, the sequence motifs utilized by this factor for specific target exon selection and its role in splicing silencing are not understood. Here, we use chemical modification footprinting to map the contact sites of CUGBP2 to GU-rich motifs closely positioned at the boundaries of the branch sites of the NI exon, and we demonstrate a mechanistic role for this specific arrangement of motifs for the regulation of branchpoint formation. General support for a branch site-perimeter-binding model is indicated by the identification of a group of novel target exons with a similar configuration of motifs that are silenced by CUGBP2. These results reveal an autoregulatory role for CUGBP2 as indicated by its direct interaction with functionally significant RNA motifs surrounding the branch sites upstream of exon 6 of the CUGBP2 transcript itself. The perimeter-binding model explains how CUGBP2 can effectively embrace the branch site region to achieve the specificity needed for the selection of exon targets and the fine-tuning of alternative splicing patterns.

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

    Directory of Open Access Journals (Sweden)

    Perrin Cécile

    2008-02-01

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

  3. N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3′ Splice Site Function in Vivo

    Science.gov (United States)

    Itani, Omar A.; Flibotte, Stephane; Dumas, Kathleen J.; Guo, Chunfang; Blumenthal, Thomas; Hu, Patrick J.

    2016-01-01

    Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3′ splice site. In the nematode Caenorhabditis elegans, 3′ splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3′ splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the –5 position of the octamer motif is the most uncommon base in C. elegans 3′ splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved –5 uridine residue in the C. elegans 3′ splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo. PMID:27172199

  4. The effect of intron location on the splicing of BmKK2 in 293T cells.

    Science.gov (United States)

    Zhijian, Cao; Chao, Dai; Dahe, Jiang; Wenxin, Li

    2006-01-01

    Previously reported results showed that the BmKK2's intron could be recognized and spliced in cultured HEK 293T cells. At the same time, a cryptic splicing site of BmKK2 gene was found in the second exon. Moreover, replacing BmKK2's intron with BmP03's intron (an artificial BmKK2-BmP03 mosaic gene) did not affect the intron's recognition and splicing, but increased the expression level of the toxin-GFP fusion protein (Cao et al., J Biochem Mol Toxicol 2006;20:1-6). In this investigation, the BmKK2's intron with 79 nucleotides length was artificially shifted from the 49th nt (the 17th Gly codon between the first base and the second base) to the 100th nt (the 34th Gly codon between the first base and the second base). Based on the constructed intron-splicing system, the results of RT-PCR and the western blotting analysis showed that the BmKK2's shifted-intron (named BmKK2-s) was not recognized and spliced correctly, but the cryptic splicing site of BmKK2 gene was still spliced in the second exon, which possibly indicated that locations of introns were very important to the recognition and splicing of introns, and splicing of introns was very much associated with the corresponding upstream and downstream exons. This result possibly provides evidence for splice-site recognition across the exons. (c) 2006 Wiley Periodicals, Inc.

  5. A splice site mutation in laminin-α2 results in a severe muscular dystrophy and growth abnormalities in zebrafish.

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    Vandana A Gupta

    Full Text Available Congenital muscular dystrophy (CMD is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2. Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2(cl501/cl501, exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8-15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.

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

    DEFF Research Database (Denmark)

    Sørensen, Annette Balle; Lund, Anders H; Kunder, Sandra

    2007-01-01

    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......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...

  7. "iSS-Hyb-mRMR": Identification of splicing sites using hybrid space of pseudo trinucleotide and pseudo tetranucleotide composition.

    Science.gov (United States)

    Iqbal, Muhammad; Hayat, Maqsood

    2016-05-01

    Gene splicing is a vital source of protein diversity. Perfectly eradication of introns and joining exons is the prominent task in eukaryotic gene expression, as exons are usually interrupted by introns. Identification of splicing sites through experimental techniques is complicated and time-consuming task. With the avalanche of genome sequences generated in the post genomic age, it remains a complicated and challenging task to develop an automatic, robust and reliable computational method for fast and effective identification of splicing sites. In this study, a hybrid model "iSS-Hyb-mRMR" is proposed for quickly and accurately identification of splicing sites. Two sample representation methods namely; pseudo trinucleotide composition (PseTNC) and pseudo tetranucleotide composition (PseTetraNC) were used to extract numerical descriptors from DNA sequences. Hybrid model was developed by concatenating PseTNC and PseTetraNC. In order to select high discriminative features, minimum redundancy maximum relevance algorithm was applied on the hybrid feature space. The performance of these feature representation methods was tested using various classification algorithms including K-nearest neighbor, probabilistic neural network, general regression neural network, and fitting network. Jackknife test was used for evaluation of its performance on two benchmark datasets S1 and S2, respectively. The predictor, proposed in the current study achieved an accuracy of 93.26%, sensitivity of 88.77%, and specificity of 97.78% for S1, and the accuracy of 94.12%, sensitivity of 87.14%, and specificity of 98.64% for S2, respectively. It is observed, that the performance of proposed model is higher than the existing methods in the literature so for; and will be fruitful in the mechanism of RNA splicing, and other research academia. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3' splice site and its cis-regulatory element: possible involvement in RNA splicing.

    Science.gov (United States)

    Jacquenet, S; Ropers, D; Bilodeau, P S; Damier, L; Mougin, A; Stoltzfus, C M; Branlant, C

    2001-01-15

    The HIV-1 transcript is alternatively spliced to over 30 different mRNAs. Whether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary structure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A4b, A4c and A5 3' splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem-loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing silencer (ESS2) acting on site A3 is located in a long irregular stem-loop structure (SLS3). Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putative SR protein binding site. The other one is adjacent to ESS2. Unexpectedly, only the 3' A residue of ESS2 was protected. The suboptimal A3 polypyrimidine tract (PPT) is base paired. Using site-directed mutagenesis and transfection of a mini-HIV-1 cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an optimized PPT. Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.

  9. Strict 3' splice site sequence requirements for U2 snRNP recruitment after U2AF binding underlie a genetic defect leading to autoimmune disease.

    Science.gov (United States)

    Corrionero, Anna; Raker, Veronica A; Izquierdo, José María; Valcárcel, Juan

    2011-03-01

    We report that the 3' splice site associated with the alternatively spliced exon 6 of the Fas receptor CD95 displays strict sequence requirements and that a mutation that disrupts this particular sequence arrangement leads to constitutive exon 6 skipping in a patient suffering from autoimmune lymphoproliferative syndrome (ALPS). Specifically, we find an absolute requirement for RCAG/G at the 3' splice site (where R represents purine, and / indicates the intron/exon boundary) and the balance between exon inclusion and skipping is exquisitely sensitive to single nucleotide variations in the uridine content of the upstream polypyrimidine (Py)-tract. Biochemical experiments revealed that the ALPS patient mutation reduces U2 snRNP recruitment to the 3' splice site region and that this effect cannot be explained by decreased interaction with the U2 snRNP Auxiliary Factor U2AF, whose 65- and 35-kDa subunits recognize the Py-tract and 3' splice site AG, respectively. The effect of the mutation, which generates a tandem of two consecutive AG dinucleotides at the 3' splice site, can be suppressed by increasing the distance between the AGs, mutating the natural 3' splice site AG or increasing the uridine content of the Py-tract at a position distal from the 3' splice site. The suppressive effects of these additional mutations correlate with increased recruitment of U2 snRNP but not with U2AF binding, again suggesting that the strict architecture of Fas intron 5 3' splice site region is tuned to regulate alternative exon inclusion through modulation of U2 snRNP assembly after U2AF binding.

  10. Possible anticipation associated with a novel splice site mutation in episodic ataxia type 2.

    Science.gov (United States)

    Choi, Kwang-Dong; Yook, Ji-Won; Kim, Min-Ji; Kim, Hyang-Sook; Park, Young-Eun; Kim, Ji Soo; Choi, Jae-Hwan; Shin, Jin-Hong; Kim, Dae-Seong

    2013-09-01

    Anticipation is a phenomenon characterized by decreasing age at onset and increasing severity of symptoms of a disease in successive generations within a pedigree. Anticipation mostly occurs in neurodegenerative diseases with expansion of unstable trinucleotide repeats. However, it has not been previously pointed out in episodic ataxia type 2 (EA2). Clinical and genetic analyses were performed in nine members from three consecutive generations of a Korean family with EA2. We performed a polymerase chain reaction (PCR)-based direct sequence analysis of all coding regions of CACNA1A using genomic DNA. The clinically affected family members showed recurrent vertigo, interictal nystagmus, and childhood epilepsy. There is a decrease in the age onset (possible genetic anticipation) in three succeeding generations of the family. Genetic analysis identified a splice site mutation (p.Val1465Glyfs13X) and normal trinucleotide repeats in CACNA1A in all clinically affected and one unaffected members. Recognizing anticipation would aid in genetic counseling in EA2.

  11. The pivotal roles of TIA proteins in 5' splice-site selection of alu exons and across evolution.

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    Nurit Gal-Mark

    2009-11-01

    Full Text Available 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 approximately 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.

  12. The pivotal roles of TIA proteins in 5' splice-site selection of alu exons and across evolution.

    Directory of Open Access Journals (Sweden)

    Nurit Gal-Mark

    2009-11-01

    Full Text Available 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 approximately 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.

  13. Splice-site mutations cause Rrp6-mediated nuclear retention of the unspliced RNAs and transcriptional down-regulation of the splicing-defective genes.

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    Andrea B Eberle

    Full Text Available BACKGROUND: Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster. We have produced S2 cells that express a human beta-globin gene with mutated splice sites in intron 2 (mut beta-globin. The transcripts encoded by the mut beta-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut beta-globin transcripts are much lower than those of wild type (wt ss-globin mRNAs transcribed from the same promoter. We have compared the expression of the mut and wt beta-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut beta-globin transcripts are processed at the 3', but the mut beta-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut beta-globin transcripts are less efficiently released from the transcription site, as shown by FISH, and this defect is restored by depletion of Rrp6 by RNAi. Furthermore, transcription of the mut beta-globin gene is significantly impaired as revealed by ChIP experiments that measure the association of the RNA polymerase II with the transcribed genes. We have also shown that the mut beta-globin gene shows reduced levels of H3K4me3. CONCLUSIONS/SIGNIFICANCE: Our results show that there are at least two surveillance responses that operate cotranscriptionally in insect cells and probably in all metazoans. One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The

  14. A New Method for Identification of Eukaryotic Gene Splice Sites%一种新的真核基因剪接位点识别方法

    Institute of Scientific and Technical Information of China (English)

    王科俊; 吕俊杰; 冯伟兴; 王鑫; 贺波

    2011-01-01

    剪接位点识别是基因组分析的关键步骤.为提高真核基因剪接位点识别的精度,提出一种融合多种信息的方法.在采用序列信息与剪接位点信号信息的基础上,增加剪接调控元件信息,并引入结构信息,针对供体位点与受体位点的不同特点,为其建立不同的识别模型.实验结果表明:该方法对剪接位点的识别具有较好的效果,其识别精度可达95%以上.%Splicing site recognition is the key step in the genome analysis. To improve the identification accuracy of eukary otic gene splicing sites, a variety of information fusion recognition method of splicing sites is proposed. Based on the using sequence information and splicing site signal information, we increased splicing regulatory element information, and proposed the structure information. By analyzing the different characteristics of donor sites and acceptor sites,donor sites identification signal model, acceptor sites identification signal model,donor sites identification sequence model, acceptor sites identification sequence model were built respectively. Our results show that the accuracy of splice site recognition is greater than 95 %, suggesting that the method has great potential to achieve a good performance for splice sites identification.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

    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.

  16. Autosomal dominant pseudohypoaldosteronism type 1 with a novel splice site mutation in MR gene

    Directory of Open Access Journals (Sweden)

    Kaito Hiroshi

    2009-11-01

    Full Text Available Abstract Background Autosomal dominant pseudohypoaldosteronism type 1 (PHA1 is a rare inherited condition that is characterized by renal resistance to aldosterone as well as salt wasting, hyperkalemia, and metabolic acidosis. Renal PHA1 is caused by mutations of the human mineralcorticoid receptor gene (MR, but it is a matter of debate whether MR mutations cause mineralcorticoid resistance via haploinsufficiency or dominant negative mechanism. It was previously reported that in a case with nonsense mutation the mutant mRNA was absent in lymphocytes because of nonsense mediated mRNA decay (NMD and therefore postulated that haploinsufficiency alone can give rise to the PHA1 phenotype in patients with truncated mutations. Methods and Results We conducted genomic DNA analysis and mRNA analysis for familial PHA1 patients extracted from lymphocytes and urinary sediments and could detect one novel splice site mutation which leads to exon skipping and frame shift result in premature termination at the transcript level. The mRNA analysis showed evidence of wild type and exon-skipped RT-PCR products. Conclusion mRNA analysis have been rarely conducted for PHA1 because kidney tissues are unavailable for this disease. However, we conducted RT-PCR analysis using mRNA extracted from urinary sediments. We could demonstrate that NMD does not fully function in kidney cells and that haploinsufficiency due to NMD with premature termination is not sufficient to give rise to the PHA1 phenotype at least in this mutation of our patient. Additional studies including mRNA analysis will be needed to identify the exact mechanism of the phenotype of PHA.

  17. Comparative in vitro and in silico analyses of variants in splicing regions of BRCA1 and BRCA2 genes and characterization of novel pathogenic mutations.

    Directory of Open Access Journals (Sweden)

    Mara Colombo

    Full Text Available Several unclassified variants (UVs have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at BRCA1 and BRCA2 splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants and the nature of aberrant transcripts. Eleven variants in BRCA1 and 8 in BRCA2, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs, 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that in silico tools can be used for selecting splice site variants for in vitro analyses. However, the latter

  18. Splicing of scorpion toxin gene BmKK2 in HEK 293T cells.

    Science.gov (United States)

    Zhijian, Cao; Chao, Dai; Shijin, Yin; Yingliang, Wu; Jiqun, Sheng; Yonggang, Sha; Wenxin, Li

    2006-01-01

    Using GFP as a reporter gene, splicing of scorpion toxin gene BmKK2 was investigated in cultured HEK 293T cells. The results of RT-PCR and western blotting showed that BmKK2's intron could be recognized and spliced in cultured HEK 293T cells. At the same time, a cryptic splicing site of BmKK2 gene was found at the 91st nucleotide site of the second exon, which is a typical form of alternative splicing. For the first time, alternative splicing would partially explain the diversity of scorpion toxins at the gene level. Moreover, replacing BmKK2's intron with BmP03's intron (an artificial BmKK2-BmP03 mosaic gene) did not affect the intron's recognition and splicing, but increased the expression of the toxin-GFP fusion protein by fluorescence imaging, which indicated that both introns may regulate the expression of toxin-GFP fusion protein. The artificial BmKK2-BmP03 mosaic gene was also spliced into two kinds of mRNA molecules, which showed that sequence of intron was not absolutely conserved. The results suggested that introns of scorpion toxin genes BmKK2 and BmP03 increase the diversity of scorpion toxins and regulate the expression of their genes. 2006 Wiley Periodicals, Inc.

  19. Intron definition and a branch site adenosine at nt 385 control RNA splicing of HPV16 E6*I and E7 expression.

    Science.gov (United States)

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

    2012-01-01

    HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5' splice sites (5' ss) and three 3' splice sites (3' ss) normally used in HPV16(+) cervical cancer and its derived cell lines. The choice of two novel alternative 5' ss (nt 221 5' ss and nt 191 5' ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5' ss and nt 409 3' ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3' ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3' ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of (91)QYNK(94) to (91)PSFW(94) displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.

  20. Intron definition and a branch site adenosine at nt 385 control RNA splicing of HPV16 E6*I and E7 expression.

    Directory of Open Access Journals (Sweden)

    Masahiko Ajiro

    Full Text Available HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5' splice sites (5' ss and three 3' splice sites (3' ss normally used in HPV16(+ cervical cancer and its derived cell lines. The choice of two novel alternative 5' ss (nt 221 5' ss and nt 191 5' ss produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI. The nt 226 5' ss and nt 409 3' ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS and an adenosine at nt 385 (underlined in the BPS as a branch site to dictate the selection of the nt 409 3' ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3' ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of (91QYNK(94 to (91PSFW(94 displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.

  1. Investigation of age-related changes in LMNA splicing and expression of progerin in human skeletal muscles

    OpenAIRE

    Luo, Yue-Bei; Mitrpant, Chalermchai; Johnsen, Russell D; Fabian, Victoria A; Fletcher, Sue; Mastaglia, Frank L.; Steve D Wilton

    2013-01-01

    Age-related changes in splice-forms of LMNA, which encodes the nuclear lamina proteins lamin A/C, have not been investigated in skeletal muscle. In the rare premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS), de novo point mutations in LMNA activate a cryptic splice site in exon 11, resulting in a 150 base deletion in LMNA mRNA and accumulation of a truncated protein isoform, progerin. The LMNA Δ150 progerin transcript has also been found in trace quantities in tissues of h...

  2. Conserved and species-specific alternative splicing in mammalian genomes

    Directory of Open Access Journals (Sweden)

    Favorov Alexander V

    2007-12-01

    Full Text Available Abstract Background Alternative splicing has been shown to be one of the major evolutionary mechanisms for protein diversification and proteome expansion, since a considerable fraction of alternative splicing events appears to be species- or lineage-specific. However, most studies were restricted to the analysis of cassette exons in pairs of genomes and did not analyze functionality of the alternative variants. Results We analyzed conservation of human alternative splice sites and cassette exons in the mouse and dog genomes. Alternative exons, especially minor-isofom ones, were shown to be less conserved than constitutive exons. Frame-shifting alternatives in the protein-coding regions are less conserved than frame-preserving ones. Similarly, the conservation of alternative sites is highest for evenly used alternatives, and higher when the distance between the sites is divisible by three. The rate of alternative-exon and site loss in mouse is slightly higher than in dog, consistent with faster evolution of the former. The evolutionary dynamics of alternative sites was shown to be consistent with the model of random activation of cryptic sites. Conclusion Consistent with other studies, our results show that minor cassette exons are less conserved than major-alternative and constitutive exons. However, our study provides evidence that this is caused not only by exon birth, but also lineage-specific loss of alternative exons and sites, and it depends on exon functionality.

  3. Molecular and Functional Effects of a Splice Site Mutation in the MYL2 Gene Associated with Cardioskeletal Myopathy and Early Cardiac Death in Infants.

    Science.gov (United States)

    Zhou, Zhiqun; Huang, Wenrui; Liang, Jingsheng; Szczesna-Cordary, Danuta

    2016-01-01

    The homozygous appearance of the intronic mutation (IVS6-1) in the MYL2 gene encoding for myosin ventricular/slow-twitch skeletal regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy and early cardiac death. The IVS6-1 (c403-1G>C) mutation resulted from a cryptic splice site in MYL2 causing a frameshift and replacement of the last 32 codons by 19 different amino acids in the RLC mutant protein. Infants who were IVS6-1(+∕+)-positive died between 4 and 6 months of age due to cardiomyopathy and heart failure. In this report we have investigated the molecular mechanism and functional consequences associated with the IVS6-1 mutation using recombinant human cardiac IVS6-1 and wild-type (WT) RLC proteins. Recombinant proteins were reconstituted into RLC-depleted porcine cardiac muscle preparations and subjected to enzymatic and functional assays. IVS6-1-RLC showed decreased binding to the myosin heavy chain (MHC) compared with WT, and IVS6-1-reconstituted myosin displayed reduced binding to actin in rigor. The IVS6-1 myosin demonstrated a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT. In stopped-flow experiments, IVS6-1 myosin showed slower kinetics of the ATP induced dissociation of the acto-myosin complex and a significantly reduced slope of the kobs-[MgATP] relationship compared to WT. In skinned porcine cardiac muscles, RLC-depleted and IVS6-1 reconstituted muscle strips displayed a significant decrease in maximal contractile force and a significantly increased Ca(2+) sensitivity, both hallmarks of hypertrophic cardiomyopathy-associated mutations in MYL2. Our results showed that the amino-acid changes in IVS6-1 were sufficient to impose significant conformational alterations in the RLC protein and trigger a series of abnormal protein-protein interactions in the cardiac muscle sarcomere. Notably, the mutation disrupted the RLC-MHC interaction and the steady

  4. 基于RNA-Seq数据识别果蝇剪接位点和可变剪接事件%Identification of Novel Splice Sites and Alternative Splicing Events in Drosophila melanogaster Using RNA-seq Data

    Institute of Scientific and Technical Information of China (English)

    何涛; 王端青; 胡亚欧; 张颖; 邵卫东; 汪莉

    2011-01-01

    Gene structure prediction is the first and most fundamental step to genome analysis and annotation. Splice site and alternative splicing (AS) prediction is particularly challenging for eukaryotes. With the Next Generation sequencing technologies, RNA-seq has been used in identification of splice site and alternative splicing. In this work, 39718 fruit fly splice sites were identified based on Drosophila melanogaster testis RNA-seq data by using Tophat software, of which 10584 were new discoveries. By different donor/acceptor splice site combinations, a computational identification method has been developed and applied to predict 8477 alternative splicing events (containing four distinct classes of AS events: alternative donor site, alternative acceptor site, intron retention and exon skipping). RT-PCR successfully validated novel alternative splicing events and new isoforms in two genes. Our result indicates that RNA-seq was not only an effective and accurate method for splice site and AS event detection, but also a new technique for deciphering molecular mechanism of RNA splicing further.%完整基因结构的预测是当前生命科学研究的一个重要基础课题,其中一个关键环节是剪接位点和各种可变剪接事件的精确识别.基于转录组测序(RNA-seq)数据,识别剪接位点和可变剪接事件是近几年随着新一代测序技术发展起来的新技术策略和方法.本工作基于黑腹果蝇睾丸RNA-seq数据,使用TopHat软件成功识别出39718个果蝇剪接位点,其中有10584个新剪接位点.同时,基于剪接位点的不同组合,针对各类型可变剪接特征开发出计算识别算法,成功识别了8477个可变剪接事件(其中新识别的可变剪接事件3922个),包括可变供体位点、可变受体位点、内含子保留和外显子缺失4种类型.RT-PCR实验验证了2个果蝇基因上新识别的可变剪接事件,发现了全新的剪接异构体.进一步表明,RNA-seq数据可有效应用于

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

    Science.gov (United States)

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

    2013-11-18

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

  6. 一种基于综合信息的剪接位点识别方法%Identification method of splice sites using comprehensive information

    Institute of Scientific and Technical Information of China (English)

    王科俊; 吕俊杰; 冯伟兴; 王鑫

    2011-01-01

    To identify splice sites more accurately and efficiently, a method to recognize splice sites based on comprehensive information was proposed. By analyzing the splicing signals, splicing sequences, secondary structures of flank sequence, different splicing factor mechanism of action and other characteristics of donor sites and acceptor sites, donor sites identification signal model, acceptor sites identification signal model, donor sites identification sequence model and acceptor sites identification sequence model were built, respectively. Then the Mfold package in Vienna soft was used to predict the most stable secondary structure of flank sequences. The traditional four-letter alphabet was converted into eight-letter alphabet sequence. The sequence-structure combination strings were used for training signal models and sequence models, and then well trained models were applied to recognize splice sites. Results show that the accuracy of splice site recognition is beyond 95%, suggesting that the method has great potential to achieve a good performance for splice sites identification.%为提高剪接位点识别的精度,提出一种基于综合信息的剪接位点识别方法.通过分析供体位点与受体位点的剪接信号、剪接序列、位点附近序列的二级结构,以及剪接因子作用过程等特征,分别为供体位点与受体位点建立信号模型和序列模型;应用Vienna软件中的Mfold包预测每个剪接位点附近序列最稳定的二级结构,将传统的四字符核酸表转化为八字符核酸表,每个序列用八字符进行描述,用结合了结构信息的序列对信号模型和序列模型进行训练学习;最后用训练好的模型进行剪接位点的识别.实验结果证明:该方法对剪接位点的识别取得了很好的效果,其识别精度可达95%以上.

  7. 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

    Energy Technology Data Exchange (ETDEWEB)

    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

    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.

  8. Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo.

    Science.gov (United States)

    Waldsich, C; Semrad, K; Schroeder, R

    1998-12-01

    The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA-protein interactions in HIV both in vivo and in vitro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td) intron in vivo. All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro. Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA. In the absence of translation, two interactions between sequences in the upstream exon and the 5' and 3' splice sites trap the pre-mRNA in splicing-incompetent conformations. Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B. Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA. Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation. This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E. coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing is caused by an alternative structural element containing a cryptic 5' splice site, which serves as a substrate for the ribozyme. Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.

  9. Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition

    Science.gov (United States)

    Kondo, Yasushi; Oubridge, Chris; van Roon, Anne-Marie M; Nagai, Kiyoshi

    2015-01-01

    U1 snRNP binds to the 5′ exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing. We present two crystal structures of engineered U1 sub-structures, which together reveal at atomic resolution an almost complete network of protein–protein and RNA-protein interactions within U1 snRNP, and show how the 5′ splice site of pre-mRNA is recognised by U1 snRNP. The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5′-end of U1 snRNA. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA. The structure, together with RNA binding assays, shows that the selection of 5′-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5′-splice sites. DOI: http://dx.doi.org/10.7554/eLife.04986.001 PMID:25555158

  10. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids

    DEFF Research Database (Denmark)

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S

    2008-01-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian...... DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent...

  11. A novel splice site mutation of CRYBA3/A1 gene associated with congenital cataract in a Chinese family

    Science.gov (United States)

    Wu, Meng-Han; Yu, Yin-Hui; Hao, Qin-Long; Gong, Xiao-Hua; Yao, Ke

    2017-01-01

    AIM To identify the disease-causing mutation responsible for the presence of congenital cataract in a Chinese family. METHODS The study recruited a four-generation Chinese pedigree affected by autosomal dominant congenital cataract (ADCC). Family history and the history of cataract extraction were recorded. Blood samples were collected from individuals for DNA extraction. Direct sequencing of congenital cataract-associated genes was performed. Single-strand conformational polymorphism and bioinformatic analysis were conducted to further study the mutation. RESULTS Direct sequencing revealed a novel splice site mutation of c.30-2 A>G in the CRYBA3/A1 gene. The mutation co-segregated within all affected individuals in the family and was not found in unaffected members or 100 unrelated normal controls. These results were further confirmed by single-strand conformational polymorphism and bioinformatic analysis using the Human Splicing Finder and MaxEnt online software and Annovar computer software. CONCLUSION c.30-2 A>G mutation of CRYBA3/A1 gene is a novel mutation and broadens the genetic spectrum of ADCC.

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

    Directory of Open Access Journals (Sweden)

    Sandra Cristea

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

  13. Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II.

    Science.gov (United States)

    Matos, Liliana; Gonçalves, Vânia; Pinto, Eugénia; Laranjeira, Francisco; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R; Pérez, Belén; Alves, Sandra

    2015-12-01

    Mucopolysaccharidosis II is a lysosomal storage disorder caused by mutations in the IDS gene, including exonic alterations associated with aberrant splicing. In the present work, cell-based splicing assays were performed to study the effects of two splicing mutations in exon 3 of IDS, i.e., c.241C>T and c.257C>T, whose presence activates a cryptic splice site in exon 3 and one in exon 8, i.e., c.1122C>T that despite being a synonymous mutation is responsible for the creation of a new splice site in exon 8 leading to a transcript shorter than usual. Mutant minigene analysis and overexpression assays revealed that SRSF2 and hnRNP E1 might be involved in the use and repression of the constitutive 3' splice site of exon 3 respectively. For the c.1122C>T the use of antisense therapy to correct the splicing defect was explored, but transfection of patient fibroblasts with antisense morpholino oligonucleotides (n=3) and a locked nucleic acid failed to abolish the abnormal transcript; indeed, it resulted in the appearance of yet another aberrant splicing product. Interestingly, the oligonucleotides transfection in control fibroblasts led to the appearance of the aberrant transcript observed in patients' cells after treatment, which shows that the oligonucleotides are masking an important cis-acting element for 5' splice site regulation of exon 8. These results highlight the importance of functional studies for understanding the pathogenic consequences of mis-splicing and highlight the difficulty in developing antisense therapies involving gene regions under complex splicing regulation.

  14. Heat induction of a novel Rad9 variant from a cryptic translation initiation site reduces mitotic commitment.

    Science.gov (United States)

    Janes, Simon; Schmidt, Ulrike; Ashour Garrido, Karim; Ney, Nadja; Concilio, Susanna; Zekri, Mohamed; Caspari, Thomas

    2012-10-01

    Exposure of human cells to heat switches the activating signal of the DNA damage checkpoint from genotoxic to temperature stress. This change reduces mitotic commitment at the expense of DNA break repair. The thermal alterations behind this switch remain elusive despite the successful use of heat to sensitise cancer cells to DNA breaks. Rad9 is a highly conserved subunit of the Rad9-Rad1-Hus1 (9-1-1) checkpoint-clamp that is loaded by Rad17 onto damaged chromatin. At the DNA, Rad9 activates the checkpoint kinases Rad3(ATR) and Chk1 to arrest cells in G2. Using Schizosaccharomyces pombe as a model eukaryote, we discovered a new variant of Rad9, Rad9-M50, whose expression is specifically induced by heat. High temperatures promote alternative translation from a cryptic initiation codon at methionine-50. This process is restricted to cycling cells and is independent of the temperature-sensing mitogen-activated protein kinase (MAPK) pathway. While full-length Rad9 delays mitosis in the presence of DNA lesions, Rad9-M50 functions in a remodelled checkpoint pathway to reduce mitotic commitment at elevated temperatures. This remodelled pathway still relies on Rad1 and Hus1, but acts independently of Rad17. Heat-induction of Rad9-M50 ensures that the kinase Chk1 remains in a hypo-phosphorylated state. Elevated temperatures specifically reverse the DNA-damage-induced modification of Chk1 in a manner dependent on Rad9-M50. Taken together, heat reprogrammes the DNA damage checkpoint at the level of Chk1 by inducing a Rad9 variant that can act outside of the canonical 9-1-1 complex.

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

    Science.gov (United States)

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

    2012-06-15

    In enology, "Brett" character refers to the wine spoilage caused by the yeast Dekkera/Brettanomyces bruxellensis and its production of volatile phenolic off-flavours. However, the spoilage potential of this yeast is strain-dependent. Therefore, a rapid and reliable recognition at the strain level is a key point to avoid serious economic losses. The present work provides an operative tool to assess the genetic intraspecific variation in this species through the use of introns as molecular targets. Firstly, the available partial D./B. bruxellensis genome sequence was investigated in order to build primers annealing to introns 5' splice site sequence (ISS). This analysis allowed the detection of a non-random vocabulary flanking the site and, exploiting this feature, the creation of specific probes for strain discrimination. Secondly, the separation of the intron splice site PCR fragments was obtained throughout the set up of a capillary electrophoresis protocol, giving a 94% repeatability threshold in our experimental conditions. The comparison of results obtained with ISS-PCR/CE versus the ones performed by mtDNA RFLP revealed that the former protocol is more discriminating and allowed a reliable identification at strain level. Actually sixty D./B. bruxellensis isolates were recognised as unique strains, showing a level of similarity below 79% and confirming the high genetic polymorphism existing within the species. Two main clusters were grouped at similarity levels of about 46% and 47%, respectively, showing a poor correlation with the geographic area of isolation. Moreover, from the evolutionary point of view, the proposed technique could determine the frequency of the genome rearrangements that can occur in D./B. bruxellesis populations. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Alternative splicing interference by xenobiotics.

    Science.gov (United States)

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

    2012-06-14

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

  17. Splicing site mutations in dentin sialophosphoprotein causing dentinogenesis imperfecta type II.

    Science.gov (United States)

    Holappa, Heidi; Nieminen, Pekka; Tolva, Liisa; Lukinmaa, Pirjo-Liisa; Alaluusua, Satu

    2006-10-01

    Dentinogenesis imperfecta (DGI) type II (OMIM # 125490) is an inherited disorder affecting dentin. Defective dentin formation results in discolored teeth that are prone to attrition and fracture. To date, several mutations have been described in the dentin sialophosphoprotein (DSPP) gene, causing DGI types II and III and dentin dysplasia type II. DSPP encodes two proteins: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Here, we describe a mutational analysis of DSPP in seven Finnish families with DGI type II. We report two mutations and five single nucleotide polymorphisms. In one family we found a mutation that has been described earlier in families with different ethnicity, while in six families we found a novel g.1194C>A (IVS2-3) transversion. Bioinformatic analysis of known DSPP mutations suggests that DGI type II is usually caused by aberration of normal splicing.

  18. A novel biallelic splice site mutation of TECTA causes moderate to severe hearing impairment in an Algerian family.

    Science.gov (United States)

    Behlouli, Asma; Bonnet, Crystel; Abdi, Samia; Hasbellaoui, Mokhtar; Boudjenah, Farid; Hardelin, Jean-Pierre; Louha, Malek; Makrelouf, Mohamed; Ammar-Khodja, Fatima; Zenati, Akila; Petit, Christine

    2016-08-01

    Congenital deafness is certainly one of the most common monogenic diseases in humans, but it is also one of the most genetically heterogeneous, which makes molecular diagnosis challenging in most cases. Whole-exome sequencing in two out of three Algerian siblings affected by recessively-inherited, moderate to severe sensorineural deafness allowed us to identify a novel splice donor site mutation (c.5272+1G > A) in the gene encoding α-tectorin, a major component of the cochlear tectorial membrane. The mutation was present at the homozygous state in the three affected siblings, and at the heterozygous state in their unaffected, consanguineous parents. To our knowledge, this is the first reported TECTA mutation leading to the DFNB21 form of hearing impairment among Maghrebian individuals suffering from congenital hearing impairment, which further illustrates the diversity of the genes involved in congenital deafness in the Maghreb.

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

    Science.gov (United States)

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

    2015-04-15

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

  20. Splicing aberrations caused by constitutional RB1 gene mutations in retinoblastoma

    Indian Academy of Sciences (India)

    Vidya Latha Parsam; Mohammed Javed Ali; Santosh G Honavar; Geeta K Vemuganti; Chitra Kannabiran

    2011-06-01

    Analysis of RB1 mRNA from blood leukocytes of patients with retinoblastoma identified the effects of mutations involving consensus splice site, exonic substitution and whole-exon deletions identified in genomic DNA of these patients. In addition, this study identified mutations in cases in which no mutations were detectable in the genomic DNA. One proband had mutation at the canonical splice site at +5 position of IVS22, and analysis of the transcripts in this family revealed skipping of exon 22 in three members of this family. In one proband, a missense substitution of c.652T > G (g.56897T > G; Leu218Val) in exon 7 led to splicing aberrations involving deletions of exons 7 and 8, suggesting the formation of a cryptic splice site. In two probands with no detectable changes in the genomic DNA upon screening of RB1 exons and flanking intronic sequences, transcripts were found to have deletions of exon 6 in one, and exons 21 and 22 in another family. In two probands, RNA analysis confirmed genomic deletions involving one or more exons. This study reveals novel effects of RB1 mutations on splicing and suggests the utility of RNA analysis as an adjunct to mutational screening of genomic DNA in retinoblastoma.

  1. Splicing modulation therapy in the treatment of genetic diseases

    Directory of Open Access Journals (Sweden)

    Arechavala-Gomeza V

    2014-12-01

    Full Text Available Virginia Arechavala-Gomeza,1 Bernard Khoo,2 Annemieke Aartsma-Rus3 1Neuromuscular Disorders Group, BioCruces Health Research Institute, Barakaldo, Bizkaia, Spain; 2Endocrinology, Division of Medicine, University College London, London, UK; 3Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands All authors contributed equally to this manuscript Abstract: Antisense-mediated splicing modulation is a tool that can be exploited in several ways to provide a potential therapy for rare genetic diseases. This approach is currently being tested in clinical trials for Duchenne muscular dystrophy and spinal muscular atrophy. The present review outlines the versatility of the approach to correct cryptic splicing, modulate alternative splicing, restore the open reading frame, and induce protein knockdown, providing examples of each. Finally, we outline a possible path forward toward the clinical application of this approach for a wide variety of inherited rare diseases. Keywords: splicing, therapy, antisense oligonucleotides, cryptic splicing, alternative splicing

  2. A SIGMAR1 splice-site mutation causes distal hereditary motor neuropathy.

    Science.gov (United States)

    Li, Xiaobo; Hu, Zhengmao; Liu, Lei; Xie, Yongzhi; Zhan, Yajing; Zi, Xiaohong; Wang, Junling; Wu, Lixiang; Xia, Kun; Tang, Beisha; Zhang, Ruxu

    2015-06-16

    To identify the underlying genetic cause in a consanguineous Chinese family segregating distal hereditary motor neuropathy (dHMN) in an autosomal recessive pattern. We used whole-exome sequencing and homozygosity mapping to detect the genetic variant in 2 affected individuals of the consanguineous Chinese family with dHMN. RNA analysis of peripheral blood leukocytes and immunofluorescence and immunoblotting of stable cell lines were performed to support the pathogenicity of the identified mutation. We identified 3 shared novel homozygous variants in 3 shared homozygous regions of the affected individuals. Sequencing of these 3 variants in family members revealed the c.151+1G>T mutation in SIGMAR1 gene, which located in homozygous region spanning approximately 5.3 Mb at chromosome 9p13.1-p13.3, segregated with the dHMN phenotype. The mutation causes an alternative splicing event and generates a transcript variant with an in-frame deletion of 60 base pairs in exon 1 (c.92_151del), and results in an internally shortened protein σ1R(31_50del). The proteasomal inhibitor treatment increased the intracellular amount of σ1R(31_50del) and led to the formation of nuclear aggregates. Stable expressing σ1R(31_50del) induced endoplasmic reticulum stress and enhanced apoptosis. The homozygous c.151+1G>T mutation in SIGMAR1 caused a novel form of autosomal recessive dHMN in a Chinese consanguineous family. Endoplasmic reticulum stress may have a role in the pathogenesis of dHMN. © 2015 American Academy of Neurology.

  3. A novel splice donor site in the gag-pol gene is required for HIV-1 RNA stability

    NARCIS (Netherlands)

    M. Lutzelberger; L.S. Reinert; A.T. Das; B. Berkhout; J. Kjems

    2006-01-01

    Productive infection and successful replication of human immunodeficiency virus 1 (HIV-1) requires the balanced expression of all viral genes. This is achieved by a combination of alternative splicing events and regulated nuclear export of viral RNA. Because viral splicing is incomplete and intron-c

  4. Novel mutations in EVC cause aberrant splicing in Ellis-van Creveld syndrome.

    Science.gov (United States)

    Shi, Lisong; Luo, Chunyan; Ahmed, Mairaj K; Attaie, Ali B; Ye, Xiaoqian

    2016-04-01

    Ellis-van Creveld syndrome (EvC) is a rare autosomal recessive disorder characterized by disproportionate chondrodysplasia, postaxial polydactyly, nail dystrophy, dental abnormalities and in a proportion of patients, congenital cardiac malformations. Weyers acrofacial dysostosis (Weyers) is another dominantly inherited disorder allelic to EvC syndrome but with milder phenotypes. Both disorders can result from loss-of-function mutations in either EVC or EVC2 gene, and phenotypes associated with the two gene mutations are clinically indistinguishable. We present here a clinical and molecular analysis of a Chinese family manifested specific features of EvC syndrome. Sequencing of both EVC and EVC2 identified two novel heterozygous splice site mutations c.384+5G>C in intron 3 and c.1465-1G>A in intron 10 in EVC, which were inherited from mother and father, respectively. In vitro minigene expression assay, RT-PCR and sequencing analysis demonstrated that c.384+5G>C mutation abolished normal splice site and created a new cryptic acceptor site within exon 4, whereas c.1465-1G>A mutation affected consensus splice junction site and resulted in full exon 11 skipping. These two aberrant pre-mRNA splicing processes both produced in-frame abnormal transcripts that possibly led to abolishment of important functional domains. To our knowledge, this is the first report of EVC mutations that cause EvC syndrome in Chinese population. Our data revealed that EVC splice site mutations altered splicing pattern and helped elucidate the pathogenesis of EvC syndrome.

  5. An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

    Energy Technology Data Exchange (ETDEWEB)

    Fanning, Sean W.; Horn, James R. (NIU)

    2014-03-05

    Conventional anti-hapten antibodies typically bind low-molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain-only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti-hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an anti-methotrexate VHH (anti-MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti-MTX VHH CDR1-3 Graft revealed a micromolar binding affinity, while the crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1-3 graft, which resulted in a dramatic 1000-fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti-MTX CDR1-4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti-MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low-molecular weight ligands with high affinity and specificity, despite their reduced interface.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2015-09-15

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

  8. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs

    Science.gov (United States)

    Zhang, Xiao-Ou; Dong, Rui; Zhang, Yang; Zhang, Jia-Lin; Luo, Zheng; Zhang, Jun; Chen, Ling-Ling; Yang, Li

    2016-01-01

    Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells. PMID:27365365

  9. The 3'-terminal exon of the family of steroid and phenol sulfotransferase genes is spliced at the N-terminal glycine of the universally conserved GXXGXXK motif that forms the sulfonate donor binding site.

    OpenAIRE

    Chiba, H; Komatsu, K.; Lee, Y.C.; Tomizuka, T; Strott, C A

    1995-01-01

    The guinea pig estrogen sulfotransferase gene has been cloned and compared to three other cloned steroid and phenol sulfotransferase genes (human estrogen sulfotransferase, human phenol sulfotransferase, and guinea pig 3 alpha-hydroxysteroid sulfotransferase). The four sulfotransferase genes demonstrate a common outstanding feature: the splice sites for their 3'-terminal exons are identically located. That is, the 3'-terminal exon splice sites involve a glycine that constitutes the N-terminal...

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

    Directory of Open Access Journals (Sweden)

    Eva M Schmidt

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

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

    Science.gov (United States)

    Gigante, Maddalena; d'Altilia, Marilena; Montemurno, Eustacchio; Diella, Sterpeta; Bruno, Francesca; Netti, Giuseppe S; Ranieri, Elena; Stallone, Giovanni; Infante, Barbara; Grandaliano, Giuseppe; Gesualdo, Loreto

    2013-03-18

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

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

    Science.gov (United States)

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

    2014-09-01

    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.

  13. A Splice Variant of Bardet-Biedl Syndrome 5 (BBS5 Protein that Is Selectively Expressed in Retina.

    Directory of Open Access Journals (Sweden)

    Susan N Bolch

    Full Text Available Bardet-Biedl syndrome is a complex ciliopathy that usually manifests with some form of retinal degeneration, amongst other ciliary-related deficiencies. One of the genetic causes of this syndrome results from a defect in Bardet-Biedl Syndrome 5 (BBS5 protein. BBS5 is one component of the BBSome, a complex of proteins that regulates the protein composition in cilia. In this study, we identify a smaller molecular mass form of BBS5 as a variant formed by alternative splicing and show that expression of this splice variant is restricted to the retina.Reverse transcription PCR from RNA was used to isolate and identify potential alternative transcripts of Bbs5. A peptide unique to the C-terminus of the BBS5 splice variant was synthesized and used to prepare antibodies that selectively recognized the BBS5 splice variant. These antibodies were used on immunoblots of tissue extracts to determine the extent of expression of the alternative transcript and on tissue slices to determine the localization of expressed protein. Pull-down of fluorescently labeled arrestin1 by immunoprecipitation of the BBS5 splice variant was performed to assess functional interaction between the two proteins.PCR from mouse retinal cDNA using Bbs5-specific primers amplified a unique cDNA that was shown to be a splice variant of BBS5 resulting from the use of cryptic splicing sites in Intron 7. The resulting transcript codes for a truncated form of the BBS5 protein with a unique 24 amino acid C-terminus, and predicted 26.5 kD molecular mass. PCR screening of RNA isolated from various ciliated tissues and immunoblots of protein extracts from these same tissues showed that this splice variant was expressed in retina, but not brain, heart, kidney, or testes. Quantitative PCR showed that the splice variant transcript is 8.9-fold (+/- 1.1-fold less abundant than the full-length transcript. In the retina, the splice variant of BBS5 appears to be most abundant in the connecting cilium

  14. The dark matter of the cancer genome: aberrations in regulatory elements, untranslated regions, splice sites, non-coding RNA and synonymous mutations.

    Science.gov (United States)

    Diederichs, Sven; Bartsch, Lorenz; Berkmann, Julia C; Fröse, Karin; Heitmann, Jana; Hoppe, Caroline; Iggena, Deetje; Jazmati, Danny; Karschnia, Philipp; Linsenmeier, Miriam; Maulhardt, Thomas; Möhrmann, Lino; Morstein, Johannes; Paffenholz, Stella V; Röpenack, Paula; Rückert, Timo; Sandig, Ludger; Schell, Maximilian; Steinmann, Anna; Voss, Gjendine; Wasmuth, Jacqueline; Weinberger, Maria E; Wullenkord, Ramona

    2016-01-01

    Cancer is a disease of the genome caused by oncogene activation and tumor suppressor gene inhibition. Deep sequencing studies including large consortia such as TCGA and ICGC identified numerous tumor-specific mutations not only in protein-coding sequences but also in non-coding sequences. Although 98% of the genome is not translated into proteins, most studies have neglected the information hidden in this "dark matter" of the genome. Malignancy-driving mutations can occur in all genetic elements outside the coding region, namely in enhancer, silencer, insulator, and promoter as well as in 5'-UTR and 3'-UTR Intron or splice site mutations can alter the splicing pattern. Moreover, cancer genomes contain mutations within non-coding RNA, such as microRNA, lncRNA, and lincRNA A synonymous mutation changes the coding region in the DNA and RNA but not the protein sequence. Importantly, oncogenes such as TERT or miR-21 as well as tumor suppressor genes such as TP53/p53, APC, BRCA1, or RB1 can be affected by these alterations. In summary, coding-independent mutations can affect gene regulation from transcription, splicing, mRNA stability to translation, and hence, this largely neglected area needs functional studies to elucidate the mechanisms underlying tumorigenesis. This review will focus on the important role and novel mechanisms of these non-coding or allegedly silent mutations in tumorigenesis.

  15. A novel splice site mutation of the arginine vasopressin-neurophysin II gene identified in a kindred with autosomal dominant familial neurohypophyseal diabetes insipidus.

    Science.gov (United States)

    Tae, Hyun-Jung; Baek, Ki-Hyun; Shim, Sun-Mi; Yoo, Soon-Jib; Kang, Moo-Il; Cha, Bong-Yun; Lee, Kwang-Woo; Son, Ho-Young; Kang, Sung-Koo

    2005-01-01

    Autosomal dominant familial neurohypophyseal diabetes insipidus is an inherited deficiency of arginine vasopressin (AVP), and this is caused by mutations in the AVP-neurophysin II (AVP-NP II) gene. Most of these mutations have been located in the signal peptide or in the NP II moiety. In the present study, we have analyzed the AVP-NP II gene in a Korean family. Clinical and genetic studies were performed on three members of the family, and on a normal healthy unrelated individual. The diagnosis of neurohypophyseal diabetes insipidus was done by performing a fluid deprivation test and a vasopressin challenge. For genetic analysis, the genomic DNA was extracted and the AVP-NP II gene was amplified by polymerase chain reaction (PCR). Clinical assessment of the affected individuals confirmed the diagnosis of neurohypophyseal diabetes insipidus. Genetic analysis of the AVP-NP II gene revealed a novel deletion mutation of a single nucleotide (guanine) within the splice acceptor site of intron 2 (IVS2 +1 delG). The affected individuals were heterozygous for this mutation. We also demonstrated through RT-PCR analysis of the mutant gene that this mutation resulted in the retention of intron 2 during pre-mRNA splicing. We concluded that a novel splicing mutation in the AVP-NP II gene causes neurohypophyseal diabetes insipidus in this family.

  16. 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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. The authors now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5[prime] splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5[prime] splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G[sup [minus]1]-to-T mutation at the 5[prime] splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. 48 refs., 5 figs.

  17. Differential 3' splice site recognition of SMN1 and SMN2 transcripts by U2AF and U2 snRNP.

    Science.gov (United States)

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

    2009-04-01

    Spinal Muscular atrophy is a prevalent genetic disease caused by mutation of the SMN1 gene, which encodes the SMN protein involved in assembly of small nuclear ribonucleoprotein (snRNP) complexes. A paralog of the gene, SMN2, cannot provide adequate levels of functional SMN because exon 7 is skipped in a significant fraction of the mature transcripts. A C to T transition located at position 6 of exon 7 is critical for the difference in exon skipping between SMN1 and SMN2. Here we report that this nucleotide difference results in increased ultraviolet light-mediated crosslinking of the splicing factor U2AF(65) with the 3' splice site of SMN1 intron 6 in HeLa nuclear extract. U2 snRNP association, analyzed by native gel electrophoresis, is also more efficient on SMN1 than on SMN2, particularly under conditions of competition, suggesting more effective use of limiting factors. Two trans-acting factors implicated in SMN regulation, SF2/ASF and hnRNP A1, promote and repress, respectively, U2 snRNP recruitment to both RNAs. Interestingly, depending on the transcript and the regulatory factor, the effects on U2 binding not always correlate with changes in U2AF(65) crosslinking. Furthermore, blocking recognition of a Tra2-beta1-dependent splicing enhancer located in exon 7 inhibits U2 snRNP recruitment without affecting U2AF(65) crosslinking. Collectively, the results suggest that both U2AF binding and other steps of U2 snRNP recruitment can be control points in SMN splicing regulation.

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

    Science.gov (United States)

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

    2015-05-01

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

  19. Effect of splice-site polymorphisms of the TMPRSS4, NPHP4 and ORCTL4 genes on their mRNA expression

    Indian Academy of Sciences (India)

    Hidetaka Yamada; Kazuya Shinmura; Toshihiro Tsuneyoshi; Haruhiko Sugimura

    2005-08-01

    Genetic polymorphisms associated with structural changes of their gene product are important in terms of their potential relation with diseases. Therefore, in this study, splice-site variants of the transmembrane serine protease gene TMPRSS4, nephronophthisis gene NPHP4, and organic-cation transporter gene ORCTL4, were selected from the dbSNP single nucleotide polymorphism database as candidates to identify genetic polymorphisms associated with a structural change in their mRNA transcripts. The allele frequencies of the TMPRSS4 c.4-7A>G, NPHP4 c.2818-2A>T, and ORCTL4 c.517-2A>C polymorphisms in a Japanese population were determined to be 0.42, 0.10, and 0.27, respectively, by PCR-SSCP analysis. Next, the effect of these polymorphisms on the mode of pre-mRNA splicing was investigated by RT-PCR analysis followed by sequencing analysis. The TMPRSS4, NPHP4, and ORCTL4 polymorphisms were associated with the production of the r.4-6_4-1ins transcript, the r.2818_2823del and r.2818_2859del transcripts, and the r.517-94_517-1ins; r.517-2a>c and r.517_620del transcripts, respectively. Since the proteins encoded by all these transcripts are associated with relatively significant structural changes in the form amino acid insertion/deletion and premature termination, their functional ability may be greatly reduced. Our demonstration of structural changes in mRNA transcripts as a result of splice-site polymorphisms implies that they may be of biological significance in certain pathological conditions.

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

    Directory of Open Access Journals (Sweden)

    Cornelius Schneider

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

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

    Science.gov (United States)

    Schneider, Cornelius; Agafonov, Dmitry E; Schmitzová, Jana; Hartmuth, Klaus; Fabrizio, Patrizia; Lührmann, Reinhard

    2015-01-01

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

  2. Promoter proximal polyadenylation sites reduce transcription activity

    DEFF Research Database (Denmark)

    Andersen, Pia Kjølhede; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site...... RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels.......Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site......, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites...

  3. The RNA Splicing Response to DNA Damage.

    Science.gov (United States)

    Shkreta, Lulzim; Chabot, Benoit

    2015-10-29

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

  4. Protein encoding genes in an ancient plant: analysis of codon usage, retained genes and splice sites in a moss, Physcomitrella patens

    Directory of Open Access Journals (Sweden)

    Lang Daniel

    2005-03-01

    Full Text Available Abstract Background The moss Physcomitrella patens is an emerging plant model system due to its high rate of homologous recombination, haploidy, simple body plan, physiological properties as well as phylogenetic position. Available EST data was clustered and assembled, and provided the basis for a genome-wide analysis of protein encoding genes. Results We have clustered and assembled Physcomitrella patens EST and CDS data in order to represent the transcriptome of this non-seed plant. Clustering of the publicly available data and subsequent prediction resulted in a total of 19,081 non-redundant ORF. Of these putative transcripts, approximately 30% have a homolog in both rice and Arabidopsis transcriptome. More than 130 transcripts are not present in seed plants but can be found in other kingdoms. These potential "retained genes" might have been lost during seed plant evolution. Functional annotation of these genes reveals unequal distribution among taxonomic groups and intriguing putative functions such as cytotoxicity and nucleic acid repair. Whereas introns in the moss are larger on average than in the seed plant Arabidopsis thaliana, position and amount of introns are approximately the same. Contrary to Arabidopsis, where CDS contain on average 44% G/C, in Physcomitrella the average G/C content is 50%. Interestingly, moss orthologs of Arabidopsis genes show a significant drift of codon fraction usage, towards the seed plant. While averaged codon bias is the same in Physcomitrella and Arabidopsis, the distribution pattern is different, with 15% of moss genes being unbiased. Species-specific, sensitive and selective splice site prediction for Physcomitrella has been developed using a dataset of 368 donor and acceptor sites, utilizing a support vector machine. The prediction accuracy is better than those achieved with tools trained on Arabidopsis data. Conclusion Analysis of the moss transcriptome displays differences in gene structure, codon

  5. A nucleotide substitution at the 5′splice site of intron 1 of rice HEADING DATE 1 (HD1) gene homolog in foxtail millet, broadly found in landraces from Europe and Asia

    Institute of Scientific and Technical Information of China (English)

    Kenji Fukunaga; Naoko Izuka; Takehiro Hachiken; Satoshi Mizuguchi; Hidemi Ito; Katsuyuki Ichitani

    2015-01-01

    We investigated genetic variation of a rice HEADING DATE 1(HD1) homolog in foxtail millet. First, we searched for a rice HD1 homolog in a foxtail millet genome sequence and designed primers to amplify the entire coding sequence of the gene. We compared full HD1 gene sequences of 11 accessions (including Yugu 1, a Chinese cultivar used for genome sequencing) from various regions in Europe and Asia, found a nucleotide substitution at a putative splice site of intron 1, and designated the accessions with the nucleotide substitution as carrying a splicing variant. We verified by RT-PCR that this single nucleotide substitution causes aberrant splicing of intron 1. We investigated the geographical distribution of the splicing variant in 480 accessions of foxtail millet from various regions of Europe and Asia and part of Africa by dCAPS and found that the splicing variant is broadly distributed in Europe and Asia. Differences of heading times between accessions with wild type allele of the HD1 gene and those with the splicing variant allele were unclear. We also investigated variation in 13 accessions of ssp. viridis, the wild ancestor, and the results suggested that the wild type is predominant in the wild ancestor.

  6. A nucleotide substitution at the 5′ splice site of intron 1 of rice HEADING DATE 1(HD1) gene homolog in foxtail millet, broadly found in landraces from Europe and Asia

    Institute of Scientific and Technical Information of China (English)

    Kenji Fukunaga; Naoko Izuka; Takehiro Hachiken; Satoshi Mizuguchi; Hidemi Ito; Katsuyuki Ichitani

    2015-01-01

    We investigated genetic variation of a rice HEADING DATE 1(HD1) homolog in foxtail millet.First, we searched for a rice HD1 homolog in a foxtail millet genome sequence and designed primers to amplify the entire coding sequence of the gene. We compared full HD1 gene sequences of 11 accessions(including Yugu 1, a Chinese cultivar used for genome sequencing) from various regions in Europe and Asia, found a nucleotide substitution at a putative splice site of intron 1, and designated the accessions with the nucleotide substitution as carrying a splicing variant. We verified by RT-PCR that this single nucleotide substitution causes aberrant splicing of intron 1. We investigated the geographical distribution of the splicing variant in 480 accessions of foxtail millet from various regions of Europe and Asia and part of Africa by d CAPS and found that the splicing variant is broadly distributed in Europe and Asia. Differences of heading times between accessions with wild type allele of the HD1 gene and those with the splicing variant allele were unclear. We also investigated variation in 13 accessions of ssp. viridis, the wild ancestor, and the results suggested that the wild type is predominant in the wild ancestor.

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

    Science.gov (United States)

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

    2015-04-01

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

  8. Dwarfism with joint laxity in Friesian horses is associated with a splice site mutation in B4GALT7.

    Science.gov (United States)

    Leegwater, Peter A; Vos-Loohuis, Manon; Ducro, Bart J; Boegheim, Iris J; van Steenbeek, Frank G; Nijman, Isaac J; Monroe, Glen R; Bastiaansen, John W M; Dibbits, Bert W; van de Goor, Leanne H; Hellinga, Ids; Back, Willem; Schurink, Anouk

    2016-10-28

    Inbreeding and population bottlenecks in the ancestry of Friesian horses has led to health issues such as dwarfism. The limbs of dwarfs are short and the ribs are protruding inwards at the costochondral junction, while the head and back appear normal. A striking feature of the condition is the flexor tendon laxity that leads to hyperextension of the fetlock joints. The growth plates of dwarfs display disorganized and thickened chondrocyte columns. The aim of this study was to identify the gene defect that causes the recessively inherited trait in Friesian horses to understand the disease process at the molecular level. We have localized the genetic cause of the dwarfism phenotype by a genome wide approach to a 3 Mb region on the p-arm of equine chromosome 14. The DNA of two dwarfs and one control Friesian horse was sequenced completely and we identified the missense mutation ECA14:g.4535550C > T that cosegregated with the phenotype in all Friesians analyzed. The mutation leads to the amino acid substitution p.(Arg17Lys) of xylosylprotein beta 1,4-galactosyltransferase 7 encoded by B4GALT7. The protein is one of the enzymes that synthesize the tetrasaccharide linker between protein and glycosaminoglycan moieties of proteoglycans of the extracellular matrix. The mutation not only affects a conserved arginine codon but also the last nucleotide of the first exon of the gene and we show that it impedes splicing of the primary transcript in cultured fibroblasts from a heterozygous horse. As a result, the level of B4GALT7 mRNA in fibroblasts from a dwarf is only 2 % compared to normal levels. Mutations in B4GALT7 in humans are associated with Ehlers-Danlos syndrome progeroid type 1 and Larsen of Reunion Island syndrome. Growth retardation and ligamentous laxity are common manifestations of these syndromes. We suggest that the identified mutation of equine B4GALT7 leads to the typical dwarfism phenotype in Friesian horses due to deficient splicing of transcripts of

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

    Directory of Open Access Journals (Sweden)

    Arnaud Sartelet

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

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

    Science.gov (United States)

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

    2016-06-15

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

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

    Science.gov (United States)

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

    2007-08-01

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

  12. Relationship Between the First Base of the Donor Splice Site of Waxy Gene Intron 1 and Amylose Content in Yunnan Indigenous Rice Varieties

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    There exists a single nucleotide polymorphism, G or T, at the first base of the donor splice site of waxy gene intron 1 in rice. In order to study the relationship between the first base of the donor splice site of waxy gene intron 1 and amylose content in rice, the one-step PCR method was used to determine whether it is G or T in 220 Yunnan indigenous rice varieties from 14 districts, 55 towns/counties of Yunnan Province, and 101 varieties of which were validated by the PCR-Acc I method. According to the G/T polymorphism, 164 rice varieties showed GG-genotype, while the other 56 fell into TT-genotype, accounting for 74.5% and 25.5% of all the test varieties, respectively. When all the rice varieties were divided into indica and japonica subspecies, it was found that 80.5% of indica rice and 67.0% of japonica rice belonged to GG-genotype. The rice varieties with GG-genotype had significantly higher amylose content (18.95% on average) than those with TT-genotype (all below 16%), but 33 rice varieties with GG-genotype still had low amylose content ranging from 3.91% to 15.93%, and most of them came from the Dai minority area in the Southwest of Yunnan Province. However, there was no significant difference in the mean amylose content of the same GG or TT genotypes between indica and japonica rice,suggesting that different genetic backgrounds, indica or japonica, had no effect on amylose content. The coefficient of correlation between the genotype and amylose content was 0.733 (P<0.01).

  13. Intronic Alus influence alternative splicing.

    Directory of Open Access Journals (Sweden)

    Galit Lev-Maor

    Full Text Available Examination of the human transcriptome reveals higher levels of RNA editing than in any other organism tested to date. This is indicative of extensive double-stranded RNA (dsRNA formation within the human transcriptome. Most of the editing sites are located in the primate-specific retrotransposed element called Alu. A large fraction of Alus are found in intronic sequences, implying extensive Alu-Alu dsRNA formation in mRNA precursors. Yet, the effect of these intronic Alus on splicing of the flanking exons is largely unknown. Here, we show that more Alus flank alternatively spliced exons than constitutively spliced ones; this is especially notable for those exons that have changed their mode of splicing from constitutive to alternative during human evolution. This implies that Alu insertions may change the mode of splicing of the flanking exons. Indeed, we demonstrate experimentally that two Alu elements that were inserted into an intron in opposite orientation undergo base-pairing, as evident by RNA editing, and affect the splicing patterns of a downstream exon, shifting it from constitutive to alternative. Our results indicate the importance of intronic Alus in influencing the splicing of flanking exons, further emphasizing the role of Alus in shaping of the human transcriptome.

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

    Science.gov (United States)

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

    2006-01-01

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

  15. A novel splice-site mutation in ALS2 establishes the diagnosis of juvenile amyotrophic lateral sclerosis in a family with early onset anarthria and generalized dystonias.

    Directory of Open Access Journals (Sweden)

    Saima Siddiqi

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

  16. A novel splice-site mutation in ALS2 establishes the diagnosis of juvenile amyotrophic lateral sclerosis in a family with early onset anarthria and generalized dystonias.

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2010-10-01

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

  18. Characterization of novel alternative splicing sites in human telomerase reverse transcriptase (hTERT: analysis of expression and mutual correlation in mRNA isoforms from normal and tumour tissues

    Directory of Open Access Journals (Sweden)

    Gaudernack Gustav

    2006-08-01

    Full Text Available Abstract Background Human telomerase reverse transcriptase (hTERT is a key component for synthesis and maintenance of telomeres on chromosome ends and is required for the continued proliferation of cells. Estimation of hTERT expression therefore has broad relevance in oncology and stem cell research. Several splicing variants of hTERT have been described whose regulated expression contributes to the control of telomerase activity. Knowledge of the different hTERT mRNA isoforms and the ability to distinguish between them is an important issue when evaluating telomerase expression. Results By establishing cDNA-clone panels from lung and colon tissues, we could map hTERT clones individually for differences in DNA sequence. This made possible the identification of novel alternatively spliced sites as well as analysis of their frequency and mutual correlation in mRNA isoforms. Ten different alternatively spliced sites were detected, of which six were novel sites resulting from alternative splicing of intron 2 or 14. The majority of hTERT cDNA clones from normal and tumour lung and colon tissues encoded truncated proteins ending close after exon 2 or 6. Conclusion The increased complexity in telomerase expression revealed here has implications for our understanding of telomerase regulation and for the choice of suitable methods for addressing hTERT expression.

  19. Cryptic exon incorporation occurs in Alzheimer's brain lacking TDP-43 inclusion but exhibiting nuclear clearance of TDP-43.

    Science.gov (United States)

    Sun, Mingkuan; Bell, William; LaClair, Katherine D; Ling, Jonathan P; Han, Heather; Kageyama, Yusuke; Pletnikova, Olga; Troncoso, Juan C; Wong, Philip C; Chen, Liam L

    2017-06-01

    Abnormal accumulation of TDP-43 into cytoplasmic or nuclear inclusions with accompanying nuclear clearance, a common pathology initially identified in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), has also been found in Alzheimer' disease (AD). TDP-43 serves as a splicing repressor of nonconserved cryptic exons and that such function is compromised in brains of ALS and FTD patients, suggesting that nuclear clearance of TDP-43 underlies its inability to repress cryptic exons. However, whether TDP-43 cytoplasmic aggregates are a prerequisite for the incorporation of cryptic exons is not known. Here, we assessed hippocampal tissues from 34 human postmortem brains including cases with confirmed diagnosis of AD neuropathologic changes along with age-matched controls. We found that cryptic exon incorporation occurred in all AD cases exhibiting TDP-43 pathology. Furthermore, incorporation of cryptic exons was observed in the hippocampus when TDP-43 inclusions was restricted only to the amygdala, the earliest stage of TDP-43 progression. Importantly, cryptic exon incorporation could be detected in AD brains lacking TDP-43 inclusion but exhibiting nuclear clearance of TDP-43. These data supports the notion that the functional consequence of nuclear depletion of TDP-43 as determined by cryptic exon incorporation likely occurs as an early event of TDP-43 proteinopathy and may have greater contribution to the pathogenesis of AD than currently appreciated. Early detection and effective repression of cryptic exons in AD patients may offer important diagnostic and therapeutic implications for this devastating illness of the elderly.

  20. Cryptic exposure to arsenic.

    Science.gov (United States)

    Rossy, Kathleen M; Janusz, Christopher A; Schwartz, Robert A

    2005-01-01

    Arsenic is an odorless, colorless and tasteless element long linked with effects on the skin and viscera. Exposure to it may be cryptic. Although human intake can occur from four forms, elemental, inorganic (trivalent and pentavalent arsenic) and organic arsenic, the trivalent inorganic arsenicals constitute the major human hazard. Arsenic usually reaches the skin from occupational, therapeutic, or environmental exposure, although it still may be employed as a poison. Occupations involving new technologies are not exempt from arsenic exposure. Its acute and chronic effects are noteworthy. Treatment options exist for arsenic-induced pathology, but prevention of toxicity remains the main focus. Vitamin and mineral supplementation may play a role in the treatment of arsenic toxicity.

  1. Cryptic exposure to arsenic

    Directory of Open Access Journals (Sweden)

    Rossy Kathleen

    2005-01-01

    Full Text Available Arsenic is an odorless, colorless and tasteless element long linked with effects on the skin and viscera. Exposure to it may be cryptic. Although human intake can occur from four forms, elemental, inorganic (trivalent and pentavalent arsenic and organic arsenic, the trivalent inorganic arsenicals constitute the major human hazard. Arsenic usually reaches the skin from occupational, therapeutic, or environmental exposure, although it still may be employed as a poison. Occupations involving new technologies are not exempt from arsenic exposure. Its acute and chronic effects are noteworthy. Treatment options exist for arsenic-induced pathology, but prevention of toxicity remains the main focus. Vitamin and mineral supplementation may play a role in the treatment of arsenic toxicity.

  2. Intronic L1 retrotransposons and nested genes cause transcriptional interference by inducing intron retention, exonization and cryptic polyadenylation.

    Directory of Open Access Journals (Sweden)

    Kristel Kaer

    Full Text Available BACKGROUND: Transcriptional interference has been recently recognized as an unexpectedly complex and mostly negative regulation of genes. Despite a relatively few studies that emerged in recent years, it has been demonstrated that a readthrough transcription derived from one gene can influence the transcription of another overlapping or nested gene. However, the molecular effects resulting from this interaction are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using in silico chromosome walking, we searched for prematurely terminated transcripts bearing signatures of intron retention or exonization of intronic sequence at their 3' ends upstream to human L1 retrotransposons, protein-coding and noncoding nested genes. We demonstrate that transcriptional interference induced by intronic L1s (or other repeated DNAs and nested genes could be characterized by intron retention, forced exonization and cryptic polyadenylation. These molecular effects were revealed from the analysis of endogenous transcripts derived from different cell lines and tissues and confirmed by the expression of three minigenes in cell culture. While intron retention and exonization were comparably observed in introns upstream to L1s, forced exonization was preferentially detected in nested genes. Transcriptional interference induced by L1 or nested genes was dependent on the presence or absence of cryptic splice sites, affected the inclusion or exclusion of the upstream exon and the use of cryptic polyadenylation signals. CONCLUSIONS/SIGNIFICANCE: Our results suggest that transcriptional interference induced by intronic L1s and nested genes could influence the transcription of the large number of genes in normal as well as in tumor tissues. Therefore, this type of interference could have a major impact on the regulation of the host gene expression.

  3. Characterization of BRCA1 and BRCA2 splicing variants: a collaborative report by ENIGMA consortium members.

    Science.gov (United States)

    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

    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

  4. Altered PLP1 splicing causes hypomyelination of early myelinating structures

    DEFF Research Database (Denmark)

    Kevelam, Sietske H; Taube, Jennifer R; van Spaendonk, Rosalina M L

    2015-01-01

    : All patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20...... for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients' fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. RESULTS...... alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved...

  5. Elements located upstream and downstream of the major splice donor site influence the ability of HIV-2 leader RNA to dimerize in vitro.

    Science.gov (United States)

    Lanchy, Jean-Marc; Rentz, Casey A; Ivanovitch, John D; Lodmell, J Stephen

    2003-03-11

    An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during budding and maturation of the viral particle. In HIV-1, a 5' leader region site termed stem-loop 1 (SL1) promotes RNA dimerization in vitro and influences dimerization in vivo. In HIV-2, two sequences promote dimerization of RNA fragments in vitro: the 5'-end of the primer-binding site (PBS) and a stem-loop region homologous to the HIV-1 SL1 sequence. Because HIV-2 RNA constructs of different lengths use these two dimerization signals disproportionately, we hypothesized that other sequences could modulate their relative utilization. Here, we characterized the influence of sequences upstream and downstream of the major splice donor site on the formation of HIV-2 RNA dimers in vitro using a variety of RNA constructs and dimerization and electrophoresis protocols. We first assayed the formation of loose or tight dimers for 1-444 and 1-561 model RNAs. Although both RNAs could form PBS-dependent loose dimers, the 1-561 RNA was unable to make SL1-dependent tight dimers. Using RNAs truncated at their 5'- and/or 3'-ends and by making compensatory base substitutions, we found that two elements interfere with the formation of SL1-dependent tight dimers. The cores of these elements are located at nucleotides 189-196 and 543-550. Our results suggest that base pairing between these sequences prevents the formation of SL1-dependent tight dimers, probably by sequestering SL1 in a stable intramolecular arrangement. Moreover, we found that nucleotides downstream of SL1 decreased the rate of tight dimerization. Interestingly, dimerization at 37 degrees C in the presence of nucleocapsid protein increased the yield of SL1-mediated tight dimerization in vitro, even in the presence of the two interfering elements, suggesting a relationship between the nucleocapsid protein and activation of the SL1 dimerization signal in vivo.

  6. Regulation of Alternative Splicing in Vivo by Overexpression of Antagonistic Splicing Factors

    Science.gov (United States)

    Caceres, Javier F.; Stamm, Stefan; Helfman, David M.; Krainer, Adrian R.

    1994-09-01

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

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

    OpenAIRE

    Bauman, John A; Kole, Ryszard

    2011-01-01

    Close to 90% of human genes are transcribed into pre-mRNA that undergoes alternative splicing, producing multiple mRNAs and proteins from single genes. This process is largely responsible for human proteome diversity, and about half of genetic disease-causing mutations affect splicing. Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense therapeutics that modify gene expression by directing pre-mRNA splice site usage. Bauman et al. investigated an SSO that upregula...

  8. Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin.

    Directory of Open Access Journals (Sweden)

    Melissa K Boles

    2009-12-01

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

  9. A splice acceptor site mutation in TaGW2-A1 increases thousand grain weight in tetraploid and hexaploid wheat through wider and longer grains.

    Science.gov (United States)

    Simmonds, James; Scott, Peter; Brinton, Jemima; Mestre, Teresa C; Bush, Max; Del Blanco, Alicia; Dubcovsky, Jorge; Uauy, Cristobal

    2016-06-01

    Across 13 experiments the gw2 - A1 mutant allele shifts grain size distribution consistently across all grains significantly increasing grain weight (6.6 %), width (2.8 %) and length (2.1 %) in tetraploid and hexaploid wheat. There is an urgent need to identify, understand and incorporate alleles that benefit yield in polyploid wheat. The rice OsGW2 gene functions as a negative regulator of grain weight and width and is homologous to the wheat TaGW2 gene. Previously it was shown that transcript levels of the A-genome homoeologue, TaGW2-A1, are negatively associated with grain width in hexaploid wheat. In this study we screened the tetraploid Kronos TILLING population to identify mutants in TaGW2-A1. We identified a G to A transition in the splice acceptor site of exon 5 which leads to mis-splicing in TaGW2-A1. We backcrossed the mutant allele into tetraploid and hexaploid wheat and generated a series of backcross derived isogenic lines which were evaluated in glasshouse and field conditions. Across 13 experiments the GW2-A1 mutant allele significantly increased thousand grain weight (6.6 %), grain width (2.8 %) and grain length (2.1 %) in tetraploid and hexaploid wheat compared to the wild type allele. In hexaploid wheat, this led to an increase in spike yield since no differences were detected for spikelet or grain number between isogenic lines. The increase in grain width and length was consistent across grains of different sizes, suggesting that the effect of the mutation is stable across the ear and within spikelets. Differences in carpel size and weight between alleles were identified as early as 5 days before anthesis, suggesting that TaGW2-A1 acts on maternal tissue before anthesis to restrict seed size. A single nucleotide polymorphism marker was developed to aid the deployment of the mutant allele into breeding programmes.

  10. Widespread alternative and aberrant splicing revealed by lariat sequencing

    Science.gov (United States)

    Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

    2015-01-01

    Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

  11. Investigation of age-related changes in LMNA splicing and expression of progerin in human skeletal muscles.

    Science.gov (United States)

    Luo, Yue-Bei; Mitrpant, Chalermchai; Johnsen, Russell D; Fabian, Victoria A; Fletcher, Sue; Mastaglia, Frank L; Wilton, Steve D

    2013-01-01

    Age-related changes in splice-forms of LMNA, which encodes the nuclear lamina proteins lamin A/C, have not been investigated in skeletal muscle. In the rare premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS), de novo point mutations in LMNA activate a cryptic splice site in exon 11, resulting in a 150 base deletion in LMNA mRNA and accumulation of a truncated protein isoform, progerin. The LMNA Δ150 progerin transcript has also been found in trace quantities in tissues of healthy people and its implication in 'natural' ageing has been proposed. We therefore investigated the expression of progerin and lamin A/C in normal human and mouse skeletal muscles of different ages. LMNA Δ150 was detected in most muscle samples from healthy individuals aged 16-71 years, but was not present in any mouse muscle samples up to the age of 18 months. Real time qPCR of human muscle samples showed that there was an age-related increase in both the full length lamin A and LMNA Δ150 transcripts, whereas their protein levels did not change significantly with age. These findings indicate that there is a basal level of mis-splicing during LMNA expression that does not change with ageing in human muscle, but at levels that do not result in increased aberrant protein. The significance of these findings in the pathophysiology of muscle ageing is uncertain and warrants further investigation.

  12. SAW: a method to identify splicing events from RNA-Seq data based on splicing fingerprints.

    Directory of Open Access Journals (Sweden)

    Kang Ning

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

  13. Seemingly neutral polymorphic variants may confer immunity to splicing-inactivating mutations

    DEFF Research Database (Denmark)

    Nielsen, Karsten Bork; Sørensen, Suzette; Cartegni, Luca

    2007-01-01

    a juxtaposed exonic splicing silencer (ESS) and is necessary to define a suboptimal 3' splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on splicing by itself, it makes splicing immune to deleterious mutations in the ESE...

  14. Accumulation of GC donor splice signals in mammals

    Directory of Open Access Journals (Sweden)

    Koonin Eugene V

    2008-07-01

    Full Text Available Abstract The GT dinucleotide in the first two intron positions is the most conserved element of the U2 donor splice signals. However, in a small fraction of donor sites, GT is replaced by GC. A substantial enrichment of GC in donor sites of alternatively spliced genes has been observed previously in human, nematode and Arabidopsis, suggesting that GC signals are important for regulation of alternative splicing. We used parsimony analysis to reconstruct evolution of donor splice sites and inferred 298 GT > GC conversion events compared to 40 GC > GT conversion events in primate and rodent genomes. Thus, there was substantive accumulation of GC donor splice sites during the evolution of mammals. Accumulation of GC sites might have been driven by selection for alternative splicing. Reviewers This article was reviewed by Jerzy Jurka and Anton Nekrutenko. For the full reviews, please go to the Reviewers' Reports section.

  15. A novel splice site mutation in the dentin sialophosphoprotein gene in a Chinese family with dentinogenesis imperfecta type II.

    Science.gov (United States)

    Wang, HaoYang; Hou, YanNing; Cui, YingXia; Huang, YuFeng; Shi, YiChao; Xia, XinYi; Lu, HongYong; Wang, YunHua; Li, XiaoJun

    2009-03-01

    Twenty-four individuals were investigated that spanned six generations in a Chinese family affected with an apparently autosomal dominant form of dentinogenesis imperfecta type II (DGI-II, OMIM #125490). All affected individuals presented with typical, clinical and radiographic features of DGI-II, but without bilateral progressive high-frequency sensorineural hearing loss. To investigate the mutated molecule, a positional candidate approach was used to determine the mutated gene in this family. Genomic DNA was obtained from 24 affected individuals, 18 unaffected relatives of the family and 50 controls. Haplotype analysis was performed using leukocyte DNA for 6 short tandem repeat (STR) markers present in chromosome 4 (D4S1534, GATA62A11, DSPP, DMP1, SPP1 and D4S1563). In the critical region between D4S1534 and DMP1, the dentin sialophosphoprotein (DSPP) gene (OMIM *125485) was considered as the strongest candidate gene. The first four exons and exon/intron boundaries of the gene were analyzed using DNA from 24 affected individuals and 18 unaffected relatives of the same family. DNA sequencing revealed a heterozygous deletion mutation in intron 2 (at positions -3 to -25), which resulted in a frameshift mutation, that changed the acceptor site sequence from CAG to AAG (IVS2-3C-->A) and may also have disrupted the branch point consensus sequence in intron 2. The mutation was found in the 24 affected individuals, but not in the 18 unaffected relatives and 50 controls. The deletion was identified by allele-specific sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. We conclude that the heterozygous deletion mutation contributed to the pathogenesis of DGI-II.

  16. A novel splice site mutation in the dentin sialophosphoprotein gene in a Chinese family with dentinogenesis imperfecta type II

    Energy Technology Data Exchange (ETDEWEB)

    Wang Haoyang [Institute of Laboratory Medicine, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002 (China); Hou Yanning [Department of Stomatology, Third Affiliated Hospital, Nanjing Traditional Chinese Medicine University, Nanjing 210001 (China); Cui Yingxia [Institute of Laboratory Medicine, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002 (China)], E-mail: cuiyx55@yahoo.com.cn; Huang Yufeng [Institute of Laboratory Medicine, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002 (China)], E-mail: huangyf@androl.cn; Shi Yichao; Xia Xinyi; Lu Hongyong; Wang Yunhua; Li Xiaojun [Institute of Laboratory Medicine, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002 (China)

    2009-03-09

    Twenty-four individuals were investigated that spanned six generations in a Chinese family affected with an apparently autosomal dominant form of dentinogenesis imperfecta type II (DGI-II, OMIM 125490). All affected individuals presented with typical, clinical and radiographic features of DGI-II, but without bilateral progressive high-frequency sensorineural hearing loss. To investigate the mutated molecule, a positional candidate approach was used to determine the mutated gene in this family. Genomic DNA was obtained from 24 affected individuals, 18 unaffected relatives of the family and 50 controls. Haplotype analysis was performed using leukocyte DNA for 6 short tandem repeat (STR) markers present in chromosome 4 (D4S1534, GATA62A11, DSPP, DMP1, SPP1 and D4S1563). In the critical region between D4S1534 and DMP1, the dentin sialophosphoprotein (DSPP) gene (OMIM *125485) was considered as the strongest candidate gene. The first four exons and exon/intron boundaries of the gene were analyzed using DNA from 24 affected individuals and 18 unaffected relatives of the same family. DNA sequencing revealed a heterozygous deletion mutation in intron 2 (at positions -3 to -25), which resulted in a frameshift mutation, that changed the acceptor site sequence from CAG to AAG (IVS2-3C{yields}A) and may also have disrupted the branch point consensus sequence in intron 2. The mutation was found in the 24 affected individuals, but not in the 18 unaffected relatives and 50 controls. The deletion was identified by allele-specific sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. We conclude that the heterozygous deletion mutation contributed to the pathogenesis of DGI-II.

  17. Intronic splicing mutations in PTCH1 cause Gorlin syndrome.

    Science.gov (United States)

    Bholah, Zaynab; Smith, Miriam J; Byers, Helen J; Miles, Emma K; Evans, D Gareth; Newman, William G

    2014-09-01

    Gorlin syndrome is an autosomal dominant disorder characterized by multiple early-onset basal cell carcinoma, odontogenic keratocysts and skeletal abnormalities. It is caused by heterozygous mutations in the tumour suppressor PTCH1. Routine clinical genetic testing, by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to confirm a clinical diagnosis of Gorlin syndrome, identifies a mutation in 60-90 % of cases. We undertook RNA analysis on lymphocytes from ten individuals diagnosed with Gorlin syndrome, but without known PTCH1 mutations by exonic sequencing or MLPA. Two altered PTCH1 transcripts were identified. Genomic DNA sequence analysis identified an intron 7 mutation c.1068-10T>A, which created a strong cryptic splice acceptor site, leading to an intronic insertion of eight bases; this is predicted to create a frameshift p.(His358Alafs*12). Secondly, a deep intronic mutation c.2561-2057A>G caused an inframe insertion of 78 intronic bases in the cDNA transcript, leading to a premature stop codon p.(Gly854fs*3). The mutations are predicted to cause loss of function of PTCH1, consistent with its tumour suppressor function. The findings indicate the importance of RNA analysis to detect intronic mutations in PTCH1 not identified by routine screening techniques.

  18. X-linked agammaglobulinemia in a 10-year-old boy with a novel non-invariant splice-site mutation in Btk gene.

    Science.gov (United States)

    Maekawa, Kota; Yamada, Masafumi; Okura, Yuka; Sato, Yasumasa; Yamada, Yutaka; Kawamura, Nobuaki; Ariga, Tadashi

    2010-04-15

    X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk). Most XLA patients have severely reduced or absent peripheral blood B cells and serum immunoglobulins, since the expression or function of Btk, critical for the maturation of B cell lineages at pro-B and pre-B cell stages, is deficient. Early and accurate diagnosis of XLA is important, since the affected patients suffer from severe and recurrent infections unless they receive intravenous immunoglobulin (IVIG) replacement therapy. However, the diagnosis of XLA is not always easy because some patients have detectable ( approximately 2%) B cells in the peripheral blood and have significant levels of serum immunoglobulins. In this study, we report on a patient who was diagnosed with XLA at the age of 10years. The diagnosis was delayed due to near-normal levels of serum immunoglobulins, although he presented with severe and recurrent bacterial infections since the age of 1year. He was demonstrated to have a novel non-invariant splice-site mutation in intron 10 (IVS10 -11C-->A) of the Btk gene, which was not detected by the standard PCR-based mutation analysis. This mutation resulted in no detectable Btk expression. This case suggests that patients suffering from severe or recurrent bacterial infection should be suspected to have XLA even though they may have significant levels of serum immunoglobulins. Furthermore, significant levels of serum immunoglobulins in XLA patients do not necessarily mean less severe phenotype.

  19. Aberrant splicing of androgenic receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Ris-Stalpers, C.; Kuiper, G.G.J.M.; Faber, P.W.; van Rooij, H.C.J.; Degenhart, H.J.; Trapman, J.; Brinkmann, A.O. (Erasmus Univ., Rotterdam (Netherlands)); Schweikert, H.U. (Univ. of Bonn (Germany)); Zegers, N.D. (Medical Biological Laboratory-Organization for Applied Scientific Research, Rijswijk (Netherlands)); Hodgins, M.B. (Glasgow Univ. (United Kingdom))

    1990-10-01

    Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symtpoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. The authors report a G {r arrow} T mutation in the splice donor site of intron 4 of the androgen receptor gene of a 46, XY subject lacking detectable androgen binding to the receptor and with the complete form of androgen insensitivity. This point mutation completely abolishes normal RNA splicing at the exon 4/intron 4 boundary and results in the activation of a cryptic splice donor site in exon 4, which leads to the deletion of 123 nucleotides from the mRNA. Translation of the mutant mRNA results in an androgen receptor protein {approx}5 kDa smaller than the wild type. This mutated androgen receptor protein was unable to bind androgens and unable to activate transcription of an androgen-regulated reporter gene construct. This mutation in the human androgen receptor gene demonstrates the importance of an intact steroid-binding domain for proper androgen receptor functioning in vivo.

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

    Science.gov (United States)

    Qi, Junlin; Su, Shihuang; Mattox, William

    2007-01-01

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

  1. Assisted transcriptome reconstruction and splicing orthology

    Directory of Open Access Journals (Sweden)

    Samuel Blanquart

    2016-11-01

    Full Text Available Abstract Background Transcriptome reconstruction, defined as the identification of all protein isoforms that may be expressed by a gene, is a notably difficult computational task. With real data, the best methods based on RNA-seq data identify barely 21 % of the expressed transcripts. While waiting for algorithms and sequencing techniques to improve — as has been strongly suggested in the literature — it is important to evaluate assisted transcriptome prediction; this is the question of how alternative transcription in one species performs as a predictor of protein isoforms in another relatively close species. Most evidence-based gene predictors use transcripts from other species to annotate a genome, but the predictive power of procedures that use exclusively transcripts from external species has never been quantified. The cornerstone of such an evaluation is the correct identification of pairs of transcripts with the same splicing patterns, called splicing orthologs. Results We propose a rigorous procedural definition of splicing orthologs, based on the identification of all ortholog pairs of splicing sites in the nucleotide sequences, and alignments at the protein level. Using our definition, we compared 24 382 human transcripts and 17 909 mouse transcripts from the highly curated CCDS database, and identified 11 122 splicing orthologs. In prediction mode, we show that human transcripts can be used to infer over 62 % of mouse protein isoforms. When restricting the predictions to transcripts known eight years ago, the percentage grows to 74 %. Using CCDS timestamped releases, we also analyze the evolution of the number of splicing orthologs over the last decade. Conclusions Alternative splicing is now recognized to play a major role in the protein diversity of eukaryotic organisms, but definitions of spliced isoform orthologs are still approximate. Here we propose a definition adapted to the subtle variations of conserved alternative

  2. 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

    Energy Technology Data Exchange (ETDEWEB)

    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

    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.

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

    Directory of Open Access Journals (Sweden)

    Claudia Tammaro

    2014-07-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  5. Discovery and analysis of evolutionarily conserved intronic splicing regulatory elements.

    Directory of Open Access Journals (Sweden)

    Gene W Yeo

    2007-05-01

    Full Text Available Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs. Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5' and 94% altered 3' splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%-45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%-50% of ISREs were enriched near alternatively spliced (AS exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events.

  6. Regional rearrangements in chromosome 15q21 cause formation of cryptic promoters for the CYP19 (aromatase) gene.

    Science.gov (United States)

    Demura, Masashi; Martin, Regina M; Shozu, Makio; Sebastian, Siby; Takayama, Kazuto; Hsu, Wei-Tong; Schultz, Roger A; Neely, Kirk; Bryant, Michael; Mendonca, Berenice B; Hanaki, Keiichi; Kanzaki, Susumu; Rhoads, David B; Misra, Madhusmita; Bulun, Serdar E

    2007-11-01

    Production of appropriate quantities of estrogen in various tissues is essential for human physiology. A single gene (CYP19), regulated via tissue-specific promoters, encodes the enzyme aromatase, which catalyzes the key step in estrogen biosynthesis. Aromatase excess syndrome is inherited as autosomal dominant and characterized by high systemic estrogen levels, short stature, prepubertal gynecomastia and testicular failure in males, and premature breast development and uterine pathology in females. The underlying genetic mechanism is poorly understood. Here, we characterize five distinct heterozygous rearrangements responsible for aromatase excess syndrome in three unrelated families and two individuals (nine patients). The constitutively active promoter of one of five ubiquitously expressed genes located within the 11.2 Mb region telomeric to the CYP19 gene in chromosome 15q21 cryptically upregulated aromatase expression in several tissues. Four distinct inversions reversed the transcriptional direction of the promoter of a gene (CGNL1, TMOD3, MAPK6 or TLN2), placing it upstream of the CYP19 coding region in the opposite strand, whereas a deletion moved the promoter of a fifth gene (DMXL2), normally transcribed from the same strand, closer to CYP19. The proximal breakpoints of inversions were located 17-185 kb upstream of the CYP19 coding region. Sequences at the breakpoints suggested that the inversions were caused by intrachromosomal nonhomologous recombination. Splicing the untranslated exon downstream of each promoter onto the identical junction upstream of the translation initiation site created CYP19 mRNA encoding functional aromatase protein. Taken together, small rearrangements may create cryptic promoters that direct inappropriate transcription of CYP19 or other critical genes.

  7. Position-dependent repression and promotion of DQB1 intron 3 splicing by GGGG motifs

    National Research Council Canada - National Science Library

    Královicová, Jana; Vorechovsky, Igor

    2006-01-01

    ...) repeats on intron 3 removal. We found that the GGG or GGGG repeats generally improved splicing of DQB1 intron 3, except for those that were adjacent to the 5' splice site where they had the opposite effect...

  8. Reversion of the Arabidopsis rpn12a-1 exon-trap mutation by an intragenic suppressor that weakens the chimeric 5’ splice site [v2; ref status: indexed, http://f1000r.es/18y

    Directory of Open Access Journals (Sweden)

    Jasmina Kurepa

    2013-06-01

    Full Text Available 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.

  9. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Roy, Scott William

    2009-01-01

    and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little...

  10. Introduction to cotranscriptional RNA splicing.

    Science.gov (United States)

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

    2014-01-01

    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.

  11. Using a minigene approach to characterize a novel splice site mutation in human F7 gene causing inherited factor VII deficiency in a Chinese pedigree.

    Science.gov (United States)

    Yu, T; Wang, X; Ding, Q; Fu, Q; Dai, J; Lu, Y; Xi, X; Wang, H

    2009-11-01

    Factor VII deficiency which transmitted as an autosomal recessive disorder is a rare haemorrhagic condition. The aim of this study was to identify the molecular genetic defect and determine its functional consequences in a Chinese pedigree with FVII deficiency. The proband was diagnosed as inherited coagulation FVII deficiency by reduced plasma levels of FVII activity (4.4%) and antigen (38.5%). All nine exons and their flanking sequence of F7 gene were amplified by polymerase chain reaction (PCR) for the proband and the PCR products were directly sequenced. The compound heterozygous mutations of F7 (NM_000131.3) c.572-1G>A and F7 (NM_000131.3) c.1165T>G; p.Cys389Gly were identified in the proband's F7 gene. To investigate the splicing patterns associated with F7 c.572-1G>A, ectopic transcripts in leucocytes of the proband were analyzed. F7 minigenes, spanning from intron 4 to intron 7 and carrying either an A or a G at position -1 of intron 5, were constructed and transiently transfected into human embryonic kidney (HEK) 293T cells, followed by RT-PCR analysis. The aberrant transcripts from the F7 c.572-1G>A mutant allele were not detected by ectopic transcription study. Sequencing of the RT-PCR products from the mutant transfectant demonstrated the production of an erroneously spliced mRNA with exon 6 skipping, whereas a normal splicing occurred in the wide type transfectant. The aberrant mRNA produced from the F7 c.572-1G>A mutant allele is responsible for the factor VII deficiency in this pedigree.

  12. spliceR

    DEFF Research Database (Denmark)

    Vitting-Seerup, Kristoffer; Porse, Bo Torben; Sandelin, Albin

    2014-01-01

    RNA-seq data is currently underutilized, in part because it is difficult to predict the functional impact of alternate transcription events. Recent software improvements in full-length transcript deconvolution prompted us to develop spliceR, an R package for classification of alternative splicing...

  13. Role of Splice Variants of Gtf2i, a Transcription Factor Localizing at Postsynaptic Sites, and Its Relation to Neuropsychiatric Diseases

    Science.gov (United States)

    Shirai, Yoshinori; Li, Weidong; Suzuki, Tatsuo

    2017-01-01

    We previously reported that various mRNAs were associated with postsynaptic density (PSD) purified from rat forebrain. Among the thousands of PSD-associated mRNAs, we highlight the biology of the general transcription factor II-I (Gtf2i) mRNA, focusing on the significance of its versatile splicing for targeting its own mRNA into dendrites, regulation of translation, and the effects of Gtf2i expression level as well as its relationship with neuropsychiatric disorders. PMID:28212274

  14. The great repression: chromatin and cryptic transcription.

    Science.gov (United States)

    Hennig, Bianca P; Fischer, Tamás

    2013-01-01

    The eukaryotic chromatin structure is essential in correctly defining transcription units. Impairing this structure can activate cryptic promoters, and lead to the accumulation of aberrant RNA transcripts. Here we discuss critical pathways that are responsible for the repression of cryptic transcription and the maintenance of genome integrity.

  15. Role of cryptic genes in microbial evolution.

    Science.gov (United States)

    Hall, B G; Yokoyama, S; Calhoun, D H

    1983-12-01

    Cryptic genes are phenotypically silent DNA sequences, not normally expressed during the life cycle of an individual. They may, however, be activated in a few individuals of a large population by mutation, recombination, insertion elements, or other genetic mechanisms. A consideration of the microbial literature concerning biochemical evolution, physiology, and taxonomy provides the basis for a hypothesis of microbial adaptation and evolution by mutational activation of cryptic genes. Evidence is presented, and a mathematical model is derived, indicating that powerful and biologically important mechanisms exist to prevent the loss of cryptic genes. We propose that cryptic genes persist as a vital element of the genetic repertoire, ready for recall by mutational activation in future generations. Cryptic genes provide a versatile endogenous genetic reservoir that enhances the adaptive potential of a species by a mechanism that is independent of genetic exchange.

  16. Learning the sequence determinants of alternative splicing from millions of random sequences.

    Science.gov (United States)

    Rosenberg, Alexander B; Patwardhan, Rupali P; Shendure, Jay; Seelig, Georg

    2015-10-22

    Most human transcripts are alternatively spliced, and many disease-causing mutations affect RNA splicing. Toward better modeling the sequence determinants of alternative splicing, we measured the splicing patterns of over two million (M) synthetic mini-genes, which include degenerate subsequences totaling over 100 M bases of variation. The massive size of these training data allowed us to improve upon current models of splicing, as well as to gain new mechanistic insights. Our results show that the vast majority of hexamer sequence motifs measurably influence splice site selection when positioned within alternative exons, with multiple motifs acting additively rather than cooperatively. Intriguingly, motifs that enhance (suppress) exon inclusion in alternative 5' splicing also enhance (suppress) exon inclusion in alternative 3' or cassette exon splicing, suggesting a universal mechanism for alternative exon recognition. Finally, our empirically trained models are highly predictive of the effects of naturally occurring variants on alternative splicing in vivo.

  17. High frequency of T cells specific for cryptic epitopes in melanoma patients

    Science.gov (United States)

    Andersen, Rikke Sick; Andersen, Sofie Ramskov; Hjortsø, Mads Duus; Lyngaa, Rikke; Idorn, Manja; Køllgård, Tania Maria; Met, Özcan; thor Straten, Per; Hadrup, Sine Reker

    2013-01-01

    A number of cytotoxic T-cell epitopes are cryptic epitopes generated from non-conventional sources. These include epitopes that are encoded by alternative open reading frames or in generally non-coding genomic regions, such as introns. We have previously observed a frequent recognition of cryptic epitopes by tumor infiltrating lymphocytes isolated from melanoma patients. Here, we show that such cryptic epitopes are more frequently recognized than antigens of the same class encoded by canonical reading frames. Furthermore, we report the presence of T cells specific for three cryptic epitopes encoded in intronic sequences, as a result of incomplete splicing, in the circulation of melanoma patients. One of these epitopes derives from antigen isolated from immunoselected melanoma 2 (AIM2), while the two others are encoded in an alternative open reading frame of an incompletely spliced form of N-acetylglucosaminyl-transferase V (GNT-V) known as NA17-A. We have detected frequent T-cell responses against AIM2 and NA17-A epitopes in the blood of melanoma patients, both prior and after one round of in vitro peptide stimulation, but not in the circulation of healthy individuals and patients with breast or renal carcinoma. In summary, our findings indicate that the T-cell reactivity against AIM2 and NA17-A in the blood of melanoma patients is extensive, suggesting that—similar to melan A (also known as MART1)—these antigens might be used for immunomonitoring or as model antigens in several clinical and preclinical settings. PMID:24073381

  18. Intronic alternative splicing regulators identified by comparative genomics in nematodes.

    Directory of Open Access Journals (Sweden)

    Jennifer L Kabat

    2006-07-01

    Full Text Available Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high-scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (TGCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis

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

    Science.gov (United States)

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

    2013-08-01

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

  20. Functional analysis of three splicing mutations identified in the PMM2 gene: toward a new therapy for congenital disorder of glycosylation type Ia.

    Science.gov (United States)

    Vega, Ana I; Pérez-Cerdá, Celia; Desviat, Lourdes R; Matthijs, Gert; Ugarte, Magdalena; Pérez, Belén

    2009-05-01

    The congenital disorders of glycosylation (CDG) are a group of diseases caused by genetic defects affecting N-glycosylation. The most prevalent form of CDG-type Ia-is caused by defects in the PMM2 gene. This work reports the study of two new nucleotide changes (c.256-1G>C and c.640-9T>G) identified in the PMM2 gene in CDG1a patients, and of a previously described deep intronic nucleotide change in intron 7 (c.640-15479C>T). Cell-based splicing assays strongly suggest that all these are disease-causing splicing mutations. The c.256-1G>C mutation was found to cause the skipping of exons 3 and 4 in fibroblast cell lines and in a minigene expression system. The c.640-9T>G mutation was found responsible for the activation of a cryptic intronic splice-site in fibroblast cell lines and in a hybrid minigene when cotransfected with certain serine/arginine-rich (SR) proteins. Finally, the deep intronic change c.640-15479C>T was found to be responsible for the activation of a pseudoexon sequence in intron 7. The use of morpholino oligonucleotides allowed the production of correctly spliced mRNA that was efficiently translated into functional and immunoreactive PMM protein. The present results suggest a novel mutation-specific approach for the treatment of this genetic disease (for which no effective treatment is yet available), and open up therapeutic possibilities for several genetic disorders in which deep intronic changes are seen.

  1. Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Thomas Julie

    2010-02-01

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

  2. Optical Fiber Fusion Splicing

    CERN Document Server

    Yablon, Andrew D

    2005-01-01

    This book is an up-to-date treatment of optical fiber fusion splicing incorporating all the recent innovations in the field. It provides a toolbox of general strategies and specific techniques that the reader can apply when optimizing fusion splices between novel fibers. It specifically addresses considerations important for fusion splicing of contemporary specialty fibers including dispersion compensating fiber, erbium-doped gain fiber, polarization maintaining fiber, and microstructured fiber. Finally, it discusses the future of optical fiber fusion splicing including silica and non-silica based optical fibers as well as the trend toward increasing automation. Whilst serving as a self-contained reference work, abundant citations from the technical literature will enable readers to readily locate primary sources.

  3. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Roy, Scott William

    2009-01-01

    Alternative splicing is tightly regulated in a spatio-temporal and quantitative manner. This regulation is achieved by a complex interplay between spliceosomal (trans) factors that bind to different sequence (cis) elements. cis-elements reside in both introns and exons and may either enhance...... of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we...... and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little...

  4. Viral interactions with components of the splicing machinery.

    Science.gov (United States)

    Meyer, F

    2016-01-01

    Eukaryotic genes are often interrupted by stretches of sequence with no protein coding potential or obvious function. After transcription, these interrupting sequences must be removed to give rise to the mature messenger RNA. This fundamental process is called RNA splicing and is achieved by complicated machinery made of protein and RNA that assembles around the RNA to be edited. Viruses also use RNA splicing to maximize their coding potential and economize on genetic space, and use clever strategies to manipulate the splicing machinery to their advantage. This article gives an overview of the splicing process and provides examples of viral strategies that make use of various components of the splicing system to promote their replicative cycle. Representative virus families have been selected to illustrate the interaction with various regulatory proteins and ribonucleoproteins. The unifying theme is fine regulation through protein-protein and protein-RNA interactions with the spliceosome components and associated factors to promote or prevent spliceosome assembly on given splice sites, in addition to a strong influence from cis-regulatory sequences on viral transcripts. Because there is an intimate coupling of splicing with the processes that direct mRNA biogenesis, a description of how these viruses couple the regulation of splicing with the retention or stability of mRNAs is also included. It seems that a unique balance of suppression and activation of splicing and nuclear export works optimally for each family of viruses.

  5. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates.

    Directory of Open Access Journals (Sweden)

    Manuel Irimia

    Full Text Available Alternative splicing is tightly regulated in a spatio-temporal and quantitative manner. This regulation is achieved by a complex interplay between spliceosomal (trans factors that bind to different sequence (cis elements. cis-elements reside in both introns and exons and may either enhance or silence splicing. Differential combinations of cis-elements allows for a huge diversity of overall splicing signals, together comprising a complex 'splicing code'. Many cis-elements have been identified, and their effects on exon inclusion levels demonstrated in reporter systems. However, the impact of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs on exon inclusion levels in human and chimpanzee. For this purpose, we compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little or no effect on splicing, and thus interspecific changes at short-time scales may primarily occur in these effectively neutral ESRs. These results underscore the difficulties of using current computational ESR prediction algorithms to identify truly functionally important motifs, and provide a cautionary tale for studies of the effect of SNPs on splicing in human disease.

  6. A novel 'splice site' HCN4 Gene mutation, c.1737+1 G>T, causes familial bradycardia, reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability.

    Science.gov (United States)

    Hategan, Lidia; Csányi, Beáta; Ördög, Balázs; Kákonyi, Kornél; Tringer, Annamária; Kiss, Orsolya; Orosz, Andrea; Sághy, László; Nagy, István; Hegedűs, Zoltán; Rudas, László; Széll, Márta; Varró, András; Forster, Tamás; Sepp, Róbert

    2017-08-15

    The most important molecular determinant of heart rate regulation in sino-atrial pacemaker cells includes hyperpolarization-activated, cyclic nucleotide-gated ion channels, the major isoform of which is encoded by the HCN4 gene. Mutations affecting the HCN4 gene are associated primarily with sick sinus syndrome. A novel c.1737+1 G>T 'splice-site' HCN4 mutation was identified in a large family with familial bradycardia which co-segregated with the disease providing a two-point LOD score of 4.87. Twelve out of the 22 investigated family members [4 males, 8 females average age 36 (SD 6) years] were considered as clinically affected (heart rateheart rates [62 (SD 8) vs. 73 (SD 8) bpm, p=0.0168) were significantly lower in carriers on 24-hour Holter recordings. Under maximum exercise test carriers achieved significantly lower heart rates than non-carrier family members, and percent heart rate reserve and percent corrected heart rate reserve were significantly lower in carriers. Applying rigorous criteria for chronotropic incompetence a higher number of carriers exhibited chronotropic incompetence. Parameters, characterizing short-term variability of heart rate (i.e. rMSSD and pNN50%) were increased in carrier family members, even after normalization for heart rate, in the 24-hour ECG recordings with the same relative increase in 5-minute recordings. The identified novel 'splice site' HCN4 gene mutation, c.1737+1 G>T, causes familial bradycardia and leads to reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability in the mutation carriers. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. A combinatorial code for splicing silencing: UAGG and GGGG motifs

    National Research Council Canada - National Science Library

    Han, Kyoungha; Yeo, Gene; An, Ping; Burge, Christopher B; Grabowski, Paula J

    2005-01-01

    .... Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5'-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19...

  8. Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro

    Directory of Open Access Journals (Sweden)

    Caples Matt

    2004-07-01

    Full Text Available Abstract Background Incorporation of exon 11 of the insulin receptor gene is both developmentally and hormonally-regulated. Previously, we have shown the presence of enhancer and silencer elements that modulate the incorporation of the small 36-nucleotide exon. In this study, we investigated the role of inherent splice site strength in the alternative splicing decision and whether recognition of the splice sites is the major determinant of exon incorporation. Results We found that mutation of the flanking sub-optimal splice sites to consensus sequences caused the exon to be constitutively spliced in-vivo. These findings are consistent with the exon-definition model for splicing. In-vitro splicing of RNA templates containing exon 11 and portions of the upstream intron recapitulated the regulation seen in-vivo. Unexpectedly, we found that the splice sites are occupied and spliceosomal complex A was assembled on all templates in-vitro irrespective of splicing efficiency. Conclusion These findings demonstrate that the exon-definition model explains alternative splicing of exon 11 in the IR gene in-vivo but not in-vitro. The in-vitro results suggest that the regulation occurs at a later step in spliceosome assembly on this exon.

  9. A novel splice-site mutation in the AAGAB gene segregates with hereditary punctate palmoplantar keratoderma and congenital dysplasia of the hip in a large family.

    Science.gov (United States)

    Eytan, O; Sarig, O; Israeli, S; Mevorah, B; Basel-Vanagaite, L; Sprecher, E

    2014-03-01

    Palmoplantar keratoderma punctata (PPKP) is a heterogeneous group of disorders characterized by hyperkeratotic papules occurring over the palms and soles during adolescence. PPKP type 1, also known as PPKP Buschke-Fischer-Brauer type, was recently found to result from mutations in the AAGAB gene, encoding the p34 protein. PPKP type 1 is usually not associated with extracutaneous features. To investigate a large family in which PPKP1 was present in association with congenital dysplasia of the hip (CDH). A combination of direct sequencing of candidate genes and reverse-transcription PCR was used to identify the molecular basis underlying the clinical features displayed by the patients. Direct sequencing showed a novel intronic mutation in AAGAB, which was found to cosegregate with PPKP and CDH throughout the family. The mutation was found to result in aberrant RNA splicing, leading to exon 4 skipping. This observation suggests either the existence of a CDH-associated gene in the vicinity of AAGAB, or a hitherto unrecognized role for p34 during skeletal development. © 2013 British Association of Dermatologists.

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

    Science.gov (United States)

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

    2016-10-09

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

  11. Genome-wide identification of zero nucleotide recursive splicing in Drosophila.

    Science.gov (United States)

    Duff, Michael O; Olson, Sara; Wei, Xintao; Garrett, Sandra C; Osman, Ahmad; Bolisetty, Mohan; Plocik, Alex; Celniker, Susan E; Graveley, Brenton R

    2015-05-21

    Recursive splicing is a process in which large introns are removed in multiple steps by re-splicing at ratchet points--5' splice sites recreated after splicing. Recursive splicing was first identified in the Drosophila Ultrabithorax (Ubx) gene and only three additional Drosophila genes have since been experimentally shown to undergo recursive splicing. Here we identify 197 zero nucleotide exon ratchet points in 130 introns of 115 Drosophila genes from total RNA sequencing data generated from developmental time points, dissected tissues and cultured cells. The sequential nature of recursive splicing was confirmed by identification of lariat introns generated by splicing to and from the ratchet points. We also show that recursive splicing is a constitutive process, that depletion of U2AF inhibits recursive splicing, and that the sequence and function of ratchet points are evolutionarily conserved in Drosophila. Finally, we identify four recursively spliced human genes, one of which is also recursively spliced in Drosophila. Together, these results indicate that recursive splicing is commonly used in Drosophila, occurs in humans, and provides insight into the mechanisms by which some large introns are removed.

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

    Directory of Open Access Journals (Sweden)

    Hadas Keren-Shaul

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

  13. Identification and characterization of the soybean IPK1 ortholog of a low phytic acid mutant reveals an exon-excluding splice-site mutation.

    Science.gov (United States)

    Yuan, Feng-Jie; Zhu, Dan-Hua; Tan, Yuan-Yuan; Dong, De-Kun; Fu, Xu-Jun; Zhu, Shen-Long; Li, Bai-Quan; Shu, Qing-Yao

    2012-11-01

    Phytic acid (myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is an important constituent of soybean meal. Since phytic acid and its mineral salts (phytates) are almost indigestible for monogastrics, their abundance in grain food/feed causes nutritional and environmental problems; interest in breeding low phytic acid has therefore increased considerably. Based on gene mapping and the characteristics of inositol polyphosphates profile in the seeds of a soybean mutant line Gm-lpa-ZC-2, the soybean ortholog of inositol 1,3,4,5,6 pentakisphosphate (InsP(5)) 2-kinase (IPK1), which transforms InsP(5) into phytic acid, was first hypothesized as the candidate gene responsible for the low phytic acid alteration in Gm-lpa-ZC-2. One IPK1 ortholog (Glyma14g07880, GmIPK1) was then identified in the mapped region on chromosome 14. Sequencing revealed a G → A point mutation in the genomic DNA sequence and the exclusion of the entire fifth exon in the cDNA sequence of GmIPK1 in Gm-lpa-ZC-2 compared with its wild-type progenitor Zhechun No. 3. The excluded exon encodes 37 amino acids that spread across two conserved IPK1 motifs. Furthermore, complete co-segregation of low phytic acid phenotype with the G → A mutation was observed in the F(2) population of ZC-lpa x Zhexiandou No. 4 (a wild-type cultivar). Put together, the G → A point mutation affected the pre-mRNA splicing and resulted in the exclusion of the fifth exon of GmIPK1 which is expected to disrupt the GmIPK1 functionality, leading to low phytic acid level in Gm-lpa-ZC-2. Gm-lpa-ZC-2, would be a good germplasm source in low phytic acid soybean breeding.

  14. Genome-wide survey of allele-specific splicing in humans

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    Scheffler Konrad

    2008-06-01

    Full Text Available Abstract 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

  15. SON controls cell-cycle progression by coordinated regulation of RNA splicing.

    Science.gov (United States)

    Ahn, Eun-Young; DeKelver, Russell C; Lo, Miao-Chia; Nguyen, Tuyet Ann; Matsuura, Shinobu; Boyapati, Anita; Pandit, Shatakshi; Fu, Xiang-Dong; Zhang, Dong-Er

    2011-04-22

    It has been suspected that cell-cycle progression might be functionally coupled with RNA processing. However, little is known about the role of the precise splicing control in cell-cycle progression. Here, we report that SON, a large Ser/Arg (SR)-related protein, is a splicing cofactor contributing to efficient splicing of cell-cycle regulators. Downregulation of SON leads to severe impairment of spindle pole separation, microtubule dynamics, and genome integrity. These molecular defects result from inadequate RNA splicing of a specific set of cell-cycle-related genes that possess weak splice sites. Furthermore, we show that SON facilitates the interaction of SR proteins with RNA polymerase II and other key spliceosome components, suggesting its function in efficient cotranscriptional RNA processing. These results reveal a mechanism for controlling cell-cycle progression through SON-dependent constitutive splicing at suboptimal splice sites, with strong implications for its role in cancer and other human diseases.

  16. The peculiarities of large intron splicing in animals.

    Science.gov (United States)

    Shepard, Samuel; McCreary, Mark; Fedorov, Alexei

    2009-11-16

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

  17. The peculiarities of large intron splicing in animals.

    Directory of Open Access Journals (Sweden)

    Samuel Shepard

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

  18. SpliceDisease database: linking RNA splicing and disease.

    Science.gov (United States)

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

    2012-01-01

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

  19. spliceR

    DEFF Research Database (Denmark)

    Vitting-Seerup, Kristoffer; Porse, Bo Torben; Sandelin, Albin Gustav;

    2014-01-01

    BACKGROUND: RNA-seq data is currently underutilized, in part because it is difficult to predict the functional impact of alternate transcription events. Recent software improvements in full-length transcript deconvolution prompted us to develop spliceR, an R package for classification of alternat...

  20. Desmin splice variants causing cardiac and skeletal myopathy.

    Science.gov (United States)

    Park, K Y; Dalakas, M C; Goebel, H H; Ferrans, V J; Semino-Mora, C; Litvak, S; Takeda, K; Goldfarb, L G

    2000-11-01

    Desmin myopathy is a hereditary or sporadic cardiac and skeletal myopathy characterised by intracytoplasmic accumulation of desmin reactive deposits in muscle cells. We have characterised novel splice site mutations in the gene desmin resulting in deletion of the entire exon 3 during the pre-mRNA splicing. Sequencing of cDNA and genomic DNA identified a heterozygous de novo A to G change at the +3 position of the splice donor site of intron 3 (IVS3+3A-->G) in a patient with sporadic skeletal and cardiac myopathy. A G to A transition at the highly conserved -1 nucleotide position of intron 2 affecting the splice acceptor site (IVS2-1G-->A) was found in an unrelated patient with a similar phenotype. Expression of genomic DNA fragments carrying the IVS3+3A-->G and IVS2-1G-->A mutations confirmed that these mutations cause exon 3 deletion. Aberrant splicing leads to an in frame deletion of 32 complete codons and is predicted to result in mutant desmin lacking 32 amino acids from the 1B segment of the alpha helical rod. Functional analysis of the mutant desmin in SW13 (vim-) cells showed aggregation of abnormal coarse clumps of desmin positive material dispersed throughout the cytoplasm. This is the first report on the pathogenic potentials of splice site mutations in the desmin gene.

  1. SPA: a probabilistic algorithm for spliced alignment.

    Directory of Open Access Journals (Sweden)

    Erik van Nimwegen

    2006-04-01

    -canonical splice site that we also find in the mouse dataset. The SPA software package is available at http://www.biozentrum.unibas.ch/personal/nimwegen/cgi-bin/spa.cgi.

  2. Designing oligo libraries taking alternative splicing into account

    Science.gov (United States)

    Shoshan, Avi; Grebinskiy, Vladimir; Magen, Avner; Scolnicov, Ariel; Fink, Eyal; Lehavi, David; Wasserman, Alon

    2001-06-01

    We have designed sequences for DNA microarrays and oligo libraries, taking alternative splicing into account. Alternative splicing is a common phenomenon, occurring in more than 25% of the human genes. In many cases, different splice variants have different functions, are expressed in different tissues or may indicate different stages of disease. When designing sequences for DNA microarrays or oligo libraries, it is very important to take into account the sequence information of all the mRNA transcripts. Therefore, when a gene has more than one transcript (as a result of alternative splicing, alternative promoter sites or alternative poly-adenylation sites), it is very important to take all of them into account in the design. We have used the LEADS transcriptome prediction system to cluster and assemble the human sequences in GenBank and design optimal oligonucleotides for all the human genes with a known mRNA sequence based on the LEADS predictions.

  3. Cloning and expression of acetylcholinesterase from Electrophorus. Splicing pattern of the 3' exons in vivo and in transfected mammalian cells.

    Science.gov (United States)

    Simon, S; Massoulié, J

    1997-12-26

    We cloned and expressed a cDNA encoding acetylcholinesterase (AChE) of type T from Electrophorus electricus organs. When expressed in COS, HEK, and Chinese hamster ovary cells, the AChET subunits generated dimers and tetramers. The cells produced more activity at 27 than at 37 degrees C. The kinetic parameters of a recombinant enzyme, produced in the yeast Pichia pastoris, were close to those of the natural AChE. Analysis of genomic clones showed that the coding sequence is interrupted by an intron that does not exist in Torpedo and differs in its location from that observed in the mouse. This intron is preceded by a sequence encoding a non-conserved 29-amino acid peptide, which does not exist in Torpedo or mammalian AChEs. According to a three-dimensional model, this non-conserved peptide is located at the surface of the protein, opposite from the entry of the catalytic gorge; its deletion did not modify the catalytic parameters. Sequence analyses and expression of various constructs showed that the gene does not contain any H exon. We also found that splicing of transcripts in mammalian cells reveals cryptic donor sites in exons and acceptor sites in introns, which do not appear to be used in vivo.

  4. Reactivation of latently infected HIV-1 viral reservoirs and correction of aberrant alternative splicing in the LMNA gene via AMPK activation: Common mechanism of action linking HIV-1 latency and Hutchinson-Gilford progeria syndrome.

    Science.gov (United States)

    Finley, Jahahreeh

    2015-09-01

    Although the use of antiretroviral therapy (ART) has proven highly effective in controlling and suppressing HIV-1 replication, the persistence of latent but replication-competent proviruses in a small subset of CD4(+) memory T cells presents significant challenges to viral eradication from infected individuals. Attempts to eliminate latent reservoirs are epitomized by the 'shock and kill' approach, a strategy involving the combinatorial usage of compounds that influence epigenetic modulation and initiation of proviral transcription. However, efficient regulation of viral pre-mRNA splicing through manipulation of host cell splicing machinery is also indispensible for HIV-1 replication. Interestingly, aberrant alternative splicing of the LMNA gene via the usage of a cryptic splice site has been shown to be the cause of most cases of Hutchinson-Gilford progeria syndrome (HGPS), a rare genetic condition characterized by an accelerated aging phenotype due to the accumulation of a truncated form of lamin A known as progerin. Recent evidence has shown that inhibition of the splicing factors ASF/SF2 (or SRSF1) and SRp55 (or SRSF6) leads to a reduction or an increase in progerin at both the mRNA and protein levels, respectively, thus altering the LMNA pre-mRNA splicing ratio. It is also well-established that during the latter stages of HIV-1 infection, an increase in the production and nuclear export of unspliced viral mRNA is indispensible for efficient HIV-1 replication and that the presence of ASF/SF2 leads to excessive viral pre-mRNA splicing and a reduction of unspliced mRNA, while the presence of SRp55 inhibits viral pre-mRNA splicing and aids in the generation and translation of unspliced HIV-1 mRNAs. The splicing-factor associated protein and putative mitochondrial chaperone p32 has also been shown to inhibit ASF/SF2, increase unspliced HIV-1 viral mRNA, and enhance mitochondrial DNA replication and oxidative phosphorylation. It is our hypothesis that activation of

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

    Science.gov (United States)

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

    2015-01-01

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

  6. Binding of a candidate splice regulator to a calcitonin-specific splice enhancer regulates calcitonin/CGRP pre-mRNA splicing.

    Science.gov (United States)

    Coleman, Timothy P; Tran, Quincy; Roesser, James R

    2003-01-27

    The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. A candidate calcitonin/CGRP splice regulator (CSR) isolated from rat brain was shown to inhibit calcitonin-specific splicing in vitro. CSR specifically binds to two regions in the calcitonin-specific exon 4 RNA previously demonstrated to function as a bipartate exonic splice enhancer (ESE). The two regions, A and B element, are necessary for inclusion of exon 4 into calcitonin mRNA. A novel RNA footprinting method based on the UV cross-linking assay was used to define the site of interaction between CSR and B element RNA. Base changes at the CSR binding site prevented CSR binding to B element RNA and CSR was unable to inhibit in vitro splicing of pre-mRNAs containing the mutated CSR binding site. When expressed in cells that normally produce predominantly CGRP mRNA, a calcitonin/CGRP gene containing the mutated CSR binding site expressed predominantly calcitonin mRNA. These observations demonstrate that CSR binding to the calcitonin-specific ESE regulates calcitonin/CGRP pre-mRNA splicing.

  7. Inference of splicing regulatory activities by sequence neighborhood analysis.

    Directory of Open Access Journals (Sweden)

    Michael B Stadler

    2006-11-01

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

  8. 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

    Energy Technology Data Exchange (ETDEWEB)

    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

    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.

  9. Discrimination, crypticity, and incipient taxa in entamoeba.

    Science.gov (United States)

    Espinosa, Avelina; Paz-Y-Miño-C, Guillermo

    2012-01-01

    Persistent difficulties in resolving clear lineages in diverging populations of prokaryotes or unicellular eukaryotes (protistan polyphyletic groups) are challenging the classical species concept. Although multiple integrated approaches would render holistic taxonomies, most phylogenetic studies are still based on single-gene or morphological traits. Such methodologies conceal natural lineages, which are considered "cryptic." The concept of species is considered artificial and inadequate to define natural populations. Social organisms display differential behaviors toward kin than to nonrelated individuals. In "social" microbes, kin discrimination has been used to help resolve crypticity. Aggregative behavior could be explored in a nonsocial protist to define phylogenetic varieties that are considered "cryptic." Two Entamoeba invadens strains, IP-1 and VK-1:NS are considered close populations of the same "species." This study demonstrates that IP-1 and VK-1:NS trophozoites aggregate only with alike members and discriminate members of different strains based on behavioral and chemical signals. Combined morphological, behavioral/chemical, and ecological studies could improve Archamoebae phylogenies and define cryptic varieties. Evolutionary processes in which selection acted continuously and cumulatively on ancestors of Entamoeba populations gave rise to chemical and behavioral signals that allowed individuals to discriminate nonpopulation members and, gradually, to the emergence of new lineages; alternative views that claim a "Designer" or "Creator" as responsible for protistan diversity are unfounded.

  10. Identification of an alternative splicing isoform of chicken Lmbr1.

    Science.gov (United States)

    Huang, Yanqun; Chen, Wen; Li, Ning; Deng, Xuemei; Kang, Xiangtao; Liu, Xiaojun

    2011-10-01

    Lmbr1 is the key candidate gene for limb development. Until now, at least five and four alternative splicing isoforms of Lmbr1 gene have been found in human and mouse, respectively. However, only two alternative splicing isoforms of this homologous gene have been reported in chicken. In the present study, one novel chicken Lmbr1 transcript variant (designated Lmbr1-1) was identified by 5' RACE and RT-PCR. Chicken Lmbr1-1 possesses one novel transcription start site different from Lmbr1-N, and was predicted to encode one 192 amino acid protein with length variation in comparison with chicken LMBR1-N protein, which was produced by 5' spliced site variation of chicken Lmbr1-N exon 10. Comparing with Lmbr1-N transcript, chicken Lmbr1-1 exhibited restricted tissue distribution of the expression. Comparative sequence analysis revealed a highly conservative intron element between chicken and mammalians from the intron 9 of chicken Lmbr1-N, indicating their possible importance as intronic elements in the regulation of alternative splicing of Lmbr1 in vertebrates. By direct PCR sequencing the exon 10 and its flanking sequences in chicken Lmbr1-N, four variation sites/haplotypes were identified from six chicken breeds. One 797A/G nonsynonymous mutation (266Arg/Gln) locating in exon 10 of chicken Lmbr1-N was predicted to affect the exon splice enhancer motif for serine/arginine-rich protein recognition. These data demonstrated that chicken Lmbr1 was alternatively spliced to generate multiple splice forms, as was the case in mammals and each of the alternative splicing isoforms might function differentially.

  11. AltTrans: Transcript pattern variants annotated for both alternative splicing and alternative polyadenylation

    Directory of Open Access Journals (Sweden)

    Lopez Fabrice

    2006-03-01

    Full Text Available Abstract Background The three major mechanisms that regulate transcript formation involve the selection of alternative sites for transcription start (TS, splicing, and polyadenylation. Currently there are efforts that collect data & annotation individually for each of these variants. It is important to take an integrated view of these data sets and to derive a data set of alternate transcripts along with consolidated annotation. We have been developing in the past computational pipelines that generate value-added data at genome-scale on individual variant types; these include AltSplice on splicing and AltPAS on polyadenylation. We now extend these pipelines and integrate the resultant data sets to facilitate an integrated view of the contributions from splicing and polyadenylation in the formation of transcript variants. Description The AltSplice pipeline examines gene-transcript alignments and delineates alternative splice events and splice patterns; this pipeline is extended as AltTrans to delineate isoform transcript patterns for each of which both introns/exons and 'terminating' polyA site are delineated; EST/mRNA sequences that qualify the transcript pattern confirm both the underlying splicing and polyadenylation. The AltPAS pipeline examines gene-transcript alignments and delineates all potential polyA sites irrespective of underlying splicing patterns. Resultant polyA sites from both AltTrans and AltPAS are merged. The generated database reports data on alternative splicing, alternative polyadenylation and the resultant alternate transcript patterns; the basal data is annotated for various biological features. The data (named as integrated AltTrans data generated for both the organisms of human and mouse is made available through the Alternate Transcript Diversity web site at http://www.ebi.ac.uk/atd/. Conclusion The reported data set presents alternate transcript patterns that are annotated for both alternative splicing and alternative

  12. The r1162 mob proteins can promote conjugative transfer from cryptic origins in the bacterial chromosome.

    Science.gov (United States)

    Meyer, Richard

    2009-03-01

    The mobilization proteins of the broad-host-range plasmid R1162 can initiate conjugative transfer of a plasmid from a 19-bp locus that is partially degenerate in sequence. Such loci are likely to appear by chance in the bacterial chromosome and could act as cryptic sites for transfer of chromosomal DNA when R1162 is present. The R1162-dependent transfer of chromosomal DNA, initiated from one such potential site in Pectobacterium atrosepticum, is shown here. A second active site was identified in Escherichia coli, where it is also shown that large amounts of DNA are transferred. This transfer probably reflects the combined activity of the multiple cryptic origins in the chromosome. Transfer of chromosomal DNA due to the presence of a plasmid in the cytoplasm describes a previously unrecognized potential for the exchange of bacterial DNA.

  13. SAGE2Splice: unmapped SAGE tags reveal novel splice junctions.

    Directory of Open Access Journals (Sweden)

    Byron Yu-Lin Kuo

    2006-04-01

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

  14. Mutual interdependence of splicing and transcription elongation.

    Science.gov (United States)

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

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

  15. Methods for Characterization of Alternative RNA Splicing.

    Science.gov (United States)

    Harvey, Samuel E; Cheng, Chonghui

    2016-01-01

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

  16. Exome Sequencing Identifies a Novel LMNA Splice-Site Mutation and Multigenic Heterozygosity of Potential Modifiers in a Family with Sick Sinus Syndrome, Dilated Cardiomyopathy, and Sudden Cardiac Death.

    Science.gov (United States)

    Zaragoza, Michael V; Fung, Lianna; Jensen, Ember; Oh, Frances; Cung, Katherine; McCarthy, Linda A; Tran, Christine K; Hoang, Van; Hakim, Simin A; Grosberg, Anna

    2016-01-01

    The goals are to understand the primary genetic mechanisms that cause Sick Sinus Syndrome and to identify potential modifiers that may result in intrafamilial variability within a multigenerational family. The proband is a 63-year-old male with a family history of individuals (>10) with sinus node dysfunction, ventricular arrhythmia, cardiomyopathy, heart failure, and sudden death. We used exome sequencing of a single individual to identify a novel LMNA mutation and demonstrated the importance of Sanger validation and family studies when evaluating candidates. After initial single-gene studies were negative, we conducted exome sequencing for the proband which produced 9 gigabases of sequencing data. Bioinformatics analysis showed 94% of the reads mapped to the reference and identified 128,563 unique variants with 108,795 (85%) located in 16,319 genes of 19,056 target genes. We discovered multiple variants in known arrhythmia, cardiomyopathy, or ion channel associated genes that may serve as potential modifiers in disease expression. To identify candidate mutations, we focused on ~2,000 variants located in 237 genes of 283 known arrhythmia, cardiomyopathy, or ion channel associated genes. We filtered the candidates to 41 variants in 33 genes using zygosity, protein impact, database searches, and clinical association. Only 21 of 41 (51%) variants were validated by Sanger sequencing. We selected nine confirmed variants with minor allele frequencies G, a novel heterozygous splice-site mutation as the primary mutation with rare or novel variants in HCN4, MYBPC3, PKP4, TMPO, TTN, DMPK and KCNJ10 as potential modifiers and a mechanism consistent with haploinsufficiency.

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

    Science.gov (United States)

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

    2015-01-01

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

  18. Exome Sequencing Identifies a Novel LMNA Splice-Site Mutation and Multigenic Heterozygosity of Potential Modifiers in a Family with Sick Sinus Syndrome, Dilated Cardiomyopathy, and Sudden Cardiac Death.

    Directory of Open Access Journals (Sweden)

    Michael V Zaragoza

    Full Text Available The goals are to understand the primary genetic mechanisms that cause Sick Sinus Syndrome and to identify potential modifiers that may result in intrafamilial variability within a multigenerational family. The proband is a 63-year-old male with a family history of individuals (>10 with sinus node dysfunction, ventricular arrhythmia, cardiomyopathy, heart failure, and sudden death. We used exome sequencing of a single individual to identify a novel LMNA mutation and demonstrated the importance of Sanger validation and family studies when evaluating candidates. After initial single-gene studies were negative, we conducted exome sequencing for the proband which produced 9 gigabases of sequencing data. Bioinformatics analysis showed 94% of the reads mapped to the reference and identified 128,563 unique variants with 108,795 (85% located in 16,319 genes of 19,056 target genes. We discovered multiple variants in known arrhythmia, cardiomyopathy, or ion channel associated genes that may serve as potential modifiers in disease expression. To identify candidate mutations, we focused on ~2,000 variants located in 237 genes of 283 known arrhythmia, cardiomyopathy, or ion channel associated genes. We filtered the candidates to 41 variants in 33 genes using zygosity, protein impact, database searches, and clinical association. Only 21 of 41 (51% variants were validated by Sanger sequencing. We selected nine confirmed variants with minor allele frequencies G, a novel heterozygous splice-site mutation as the primary mutation with rare or novel variants in HCN4, MYBPC3, PKP4, TMPO, TTN, DMPK and KCNJ10 as potential modifiers and a mechanism consistent with haploinsufficiency.

  19. Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

    Directory of Open Access Journals (Sweden)

    Mario Gustavo Mayer

    2012-06-01

    Full Text Available The addition of a capped mini-exon [spliced leader (SL] through trans-splicing is essential for the maturation of RNA polymerase (pol II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1 in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin, we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

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

    Science.gov (United States)

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

    2016-02-01

    RNA splicing plays important roles in transcriptome and proteome diversity. Herein, we describe the use of a nanoplasmonic system that unveils RNA folding and assembly during pre-mRNA splicing wherein the quantification of mRNA splice variants is not taken into account. With a couple of SERS-probes and plasmonic probes binding at the boundary sites of exon-2/intron-2 and intron-2/exon-3 of the pre-mature RNA of the β-globin gene, the splicing process brings the probes into the plasmonic bands. For plasmonic probes, a plasmon shift increase of ~29 nm, corresponding to intron removal and exon-2 and exon-3 connection to form the mRNA molecule, is measured by plasmonic coupling. The increased scattering intensity and surface-enhanced Raman scattering (SERS) fingerprinting reveal the clear dynamics of pre-mRNA splicing. Moreover, a time-resolved experiment of individual RNA molecules exhibited a successful splicing and an inhibited splicing event by 33 μM biflavonoid isoginkgetin, a general inhibitor of RNA splicing. The results suggest that the RNA splicing is successfully monitored with the nanoplasmonic system. Thus, this platform can be useful for studying RNA nanotechnology, biomolecular folding, alternative splicing, and maturation of microRNA.

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

    National Research Council Canada - National Science Library

    Schneider, Cornelius; Agafonov, Dmitry E; Schmitzová, Jana; Hartmuth, Klaus; Fabrizio, Patrizia; Lührmann, Reinhard

    2015-01-01

    ..., Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site...

  2. Internal ribosome entry site mediates protein synthesis in yeast Pichia pastoris.

    Science.gov (United States)

    Liang, Shuli; Lin, Ying; Li, Cheng; Ye, Yanrui

    2012-05-01

    The imitation of translation, as mediated by internal ribosome entry sites, has not yet been reported in Pichia pastoris. An IRES element from Saccharomyces cerevisiae was demonstrated to direct the translation of a dicistronic mRNA in P. pastoris. The 5′-untranslated region of GPR1 mRNA, termed GPR, was cloned into a dual reporter construct containing an upstream Rhizomucor miehei lipase (RML) and a downstream β-galactosidase gene (lacZ) from Escherichia coli BL21. After being transformed into P. pastoris, the RML gene and lacZ were simultaneously expressed. The possibility of DNA rearrangement, spurious splicing, or cryptic promoter in the GPR sequence were eliminated, indicating that expression of a second ORF was IRES-dependent. These findings strongly suggested that the IRES-dependent translation initiation mechanism is conserved in P. pastoris and provides a useful means to express multiple genes simultaneously.

  3. Differential responses of cryptic bat species to the urban landscape.

    Science.gov (United States)

    Lintott, Paul R; Barlow, Kate; Bunnefeld, Nils; Briggs, Philip; Gajas Roig, Clara; Park, Kirsty J

    2016-04-01

    Urbanization is a key global driver in the modification of land use and has been linked to population declines even in widespread and relatively common species. Cities comprise a complex assortment of habitat types yet we know relatively little about the effects of their composition and spatial configuration on species distribution. Although many bat species exploit human resources, the majority of species are negatively impacted by urbanization. Here, we use data from the National Bat Monitoring Programme, a long-running citizen science scheme, to assess how two cryptic European bat species respond to the urban landscape. A total of 124 × 1 km(2) sites throughout Britain were surveyed. The landscape surrounding each site was mapped and classified into discrete biotope types (e.g., woodland). Generalized linear models were used to assess differences in the response to the urban environment between the two species, and which landscape factors were associated with the distributions of P. pipistrellus and P. pygmaeus. The relative prevalence of P. pygmaeus compared to P. pipistrellus was greater in urban landscapes with a higher density of rivers and lakes, whereas P. pipistrellus was frequently detected in landscapes comprising a high proportion of green space (e.g., parklands). Although P. pipistrellus is thought to be well adapted to the urban landscape, we found a strong negative response to urbanization at a relatively local scale (1 km), whilst P. pygmaeus was detected more regularly in wooded urban landscapes containing freshwater. These results show differential habitat use at a landscape scale of two morphologically similar species, indicating that cryptic species may respond differently to anthropogenic disturbance. Even species considered relatively common and well adapted to the urban landscape may respond negatively to the built environment highlighting the future challenges involved in maintaining biodiversity within an increasingly urbanized

  4. Functional splicing network reveals extensive regulatory potential of the core spliceosomal machinery.

    Science.gov (United States)

    Papasaikas, Panagiotis; Tejedor, J Ramón; Vigevani, Luisa; Valcárcel, Juan

    2015-01-08

    Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions. The network accurately captures known physical and functional associations and identifies new ones, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during spliceosome assembly. In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF, and on targets of antitumor drugs, and can be widely used to identify mechanisms of splicing regulation.

  5. Strategic performance management evaluation for the Navy's SPLICE local area networks

    OpenAIRE

    Blankenship, David D.

    1985-01-01

    Approved for public release; distribution is unlimited This thesis investigates those aspects of network performance evaluation thought to pertain specifically to strategic performance management evaluation of the Navy's Stock Point Logistics Integrated Communications Environment (SPLICE) local area networks at stock point and inventory control point sites- Background is provided concerning the SPLICE Project, strategic management, computer performance evaluation tools...

  6. Exonic splicing regulatory elements skew synonymous codon usage near intron-exon boundaries in mammals.

    NARCIS (Netherlands)

    Parmley, J.L.; Hurst, L.D.

    2007-01-01

    In mammals there is a bias in amino acid usage near splice sites that is explained, in large part, by the high density of exonic splicing enhancers (ESEs) in these regions. Is there a similar bias for the relative use of synonymous codons, and can any such bias be predicted by their abundance in ESE

  7. Splicing factors SF1 and U2AF associate in extraspliceosomal complexes

    NARCIS (Netherlands)

    Rino, J.; Desterro, J.M.P.; Pacheco, T.R.; Gadella (jr.), T.W.J.; Carmo-Fonseca, M.

    2008-01-01

    Splicing factors SF1 and U2AF associate cooperatively with pre-mRNA and play a crucial role in 3' splice site recognition during early steps of spliceosome assembly. Formation of the active spliceosome subsequently displaces SF1 in a remodeling process that stabilizes the association of U2 snRNP

  8. Activation and repression functions of an SR splicing regulator depend on exonic versus intronic-binding position.

    Science.gov (United States)

    Shen, Manli; Mattox, William

    2012-01-01

    SR proteins and related factors play widespread roles in alternative pre-mRNA splicing and are known to promote splice site recognition through their Arg-Ser-rich effector domains. However, binding of SR regulators to some targets results in repression of splice sites through a distinct mechanism. Here, we investigate how activated and repressed targets of the Drosophila SR regulator Transformer2 elicit its differing effects on splicing. We find that, like activation, repression affects early steps in the recognition of splice sites and spliceosome assembly. Repositioning of regulatory elements reveals that Tra2 complexes that normally repress splicing from intronic positions activate splicing when located in an exon. Protein tethering experiments demonstrate that this position dependence is an intrinsic property of Tra2 and further show that repression and activation are mediated by separate effector domains of this protein. When other Drosophila SR factors (SF2 and Rbp1) that activate splicing from exonic positions were tethered intronically they failed to either activate or repress splicing. Interestingly, both activities of Tra2 favor the exonic identity of the RNA sequences that encompass its binding sites. This suggests a model in which these two opposite functions act in concert to define both the position and extent of alternatively spliced exons.

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

    Science.gov (United States)

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

    2015-01-01

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

  10. Single exon mutation in arylsulfatase A gene has two effects: loss of enzyme activity and aberrant splicing.

    Science.gov (United States)

    Hasegawa, Y; Kawame, H; Ida, H; Ohashi, T; Eto, Y

    1994-04-01

    The arylsulfatase A gene of a Japanese patient who has the juvenile form of metachromatic leukodystrophy, and who has been previously reported as a heterozygote of the 1070A mutation, was investigated. Nucleotide sequence analysis revealed the presence of a previously unreported C-to-T substitution (designated 2330T), 22 nucleotides downstream from the exon 8 splice acceptor site. Although the 2330T mutation itself results in a single amino acid substitution of Thr409 by Ile, the analysis of the patient's cDNA fragments amplified by the reverse transcription-polymerase chain reaction revealed that transcripts of the 2330T allele were spliced both normally and aberrantly. The aberrant splicing produced a 27-nucleotide deletion from the usual exon 8 splice acceptor site. These results indicate that the new mutation is a rare case of an exon mutation affecting splice site selection. The mechanism of this aberrant pre-mRNA splicing is discussed.

  11. Sec16 alternative splicing dynamically controls COPII transport efficiency.

    Science.gov (United States)

    Wilhelmi, Ilka; Kanski, Regina; Neumann, Alexander; Herdt, Olga; Hoff, Florian; Jacob, Ralf; Preußner, Marco; Heyd, Florian

    2016-08-05

    The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. Here we show that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, we show that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. Our work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments.

  12. Mutations in the small subunit of the Drosophila U2AF splicing factor cause lethality and developmental defects

    NARCIS (Netherlands)

    D.Z. Rudner (David); R. Kanaar (Roland); K.S. Breger (Kevin); D.C. Rio (Donald)

    1996-01-01

    textabstractThe essential eukaryotic pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is required to specify the 3' splice site at an early step in spliceosome assembly. U2AF binds site-specifically to the intron polypyrimidine tract and recruits U2 small nuclear

  13. Handbook of knotting and splicing

    CERN Document Server

    Hasluck, Paul N

    2005-01-01

    Clearly written and amply illustrated with 208 figures, this classic guide ranges from simple and useful knots to complex varieties. Additional topics include rope splicing, working cordage, hammock making, more.

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

    Science.gov (United States)

    Kwon, Young-Ju; Park, Mi-Jeong; Kim, Sang-Gyu; Baldwin, Ian T; Park, Chung-Mo

    2014-05-19

    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. 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. 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

  15. RRM domain of Arabidopsis splicing factor SF1 is important for pre-mRNA splicing of a specific set of genes

    KAUST Repository

    Lee, Keh Chien

    2017-04-11

    The RNA recognition motif of Arabidopsis splicing factor SF1 affects the alternative splicing of FLOWERING LOCUS M pre-mRNA and a heat shock transcription factor HsfA2 pre-mRNA. Splicing factor 1 (SF1) plays a crucial role in 3\\' splice site recognition by binding directly to the intron branch point. Although plant SF1 proteins possess an RNA recognition motif (RRM) domain that is absent in its fungal and metazoan counterparts, the role of the RRM domain in SF1 function has not been characterized. Here, we show that the RRM domain differentially affects the full function of the Arabidopsis thaliana AtSF1 protein under different experimental conditions. For example, the deletion of RRM domain influences AtSF1-mediated control of flowering time, but not the abscisic acid sensitivity response during seed germination. The alternative splicing of FLOWERING LOCUS M (FLM) pre-mRNA is involved in flowering time control. We found that the RRM domain of AtSF1 protein alters the production of alternatively spliced FLM-β transcripts. We also found that the RRM domain affects the alternative splicing of a heat shock transcription factor HsfA2 pre-mRNA, thereby mediating the heat stress response. Taken together, our results suggest the importance of RRM domain for AtSF1-mediated alternative splicing of a subset of genes involved in the regulation of flowering and adaptation to heat stress.

  16. Restoration of correct splicing in IVSI-110 mutation of β-globin gene with antisense oligonucleotides: implications and applications in functional assay development

    Directory of Open Access Journals (Sweden)

    Sima Mansoori Derakhshan

    2017-06-01

    Full Text Available Objective(s: The use of antisense oligonucleotides (AOs to restore normal splicing by blocking the recognition of aberrant splice sites by the spliceosome represents an innovative means of potentially controlling certain inherited disorders affected by aberrant splicing. Selection of the appropriate target site is essential in the success of an AO therapy. In this study, in search for a splice model system to facilitate the evaluation of AOs to redirect defective splicing of IVSI-110 β-globin intron, an EGFP-based IVSI-110 specific cellular reporter assay system has been developed and a number of AOs were tested in this cellular splicing assay. Materials and Methods: A recombinant plasmid (pEGFP/I-110 carrying the EGFP gene interrupted by a mutated human β-globin intron 1 (IVSI-110 was developed and transfected into K562 cells. A number of AOs with a 2’-O-methyl oligoribonucleotide (2’-O-Me backbone system were systematically tested in this cellular splicing assay. Results: The mutation in the intron causes aberrant splicing of EGFP pre-mRNA, preventing translation of EGFP; however, treatment of the cells with specific concentration of a sequence specific 2’-O-Me AO targeted to the aberrant splice site induced correct splicing and resulted in restoring of EGFP activity. Conclusion: This cellular splicing assay provides a novel functional assay system in assessing the cellular delivery efficiency of AOs and therapeutic effect of AOs in restoration of aberrant splicing.

  17. ParSplice, Version 1

    Energy Technology Data Exchange (ETDEWEB)

    2017-01-05

    The ParSplice code implements the Parallel Trajectory Splicing algorithm described in [1]. This method is part of the Accelerated Molecular Dynamics family of techniques developed in Los Alamos National Laboratory over the last 16 years. These methods aim at generating high-quality trajectories of ensembles of atoms in materials. ParSplice uses multiple independent replicas of the system in order to parallelize the generation of such trajectories in the time domain, enabling simulations of systems of modest size over very long timescales. ParSplice includes capabilities to store configurations of the system, to generate and distribute tasks across a large number of processors, and to harvest the results of these tasks to generate long trajectories. ParSplice is a management layer that orchestrate large number of calculations, but it does not perform the actual molecular dynamics itself; this is done by external molecular dynamics engines. [1] Danny Perez, Ekin D Cubuk, Amos Waterland, Efthimios Kaxiras, Arthur F Voter, Long-time dynamics through parallel trajectory splicing, Journal of chemical theory and computation 12, 18 (2015)

  18. A method of predicting changes in human gene splicing induced by genetic variants in context of cis-acting elements

    Directory of Open Access Journals (Sweden)

    Hicks Chindo

    2010-01-01

    Full Text Available Abstract Background Polymorphic variants and mutations disrupting canonical splicing isoforms are among the leading causes of human hereditary disorders. While there is a substantial evidence of aberrant splicing causing Mendelian diseases, the implication of such events in multi-genic disorders is yet to be well understood. We have developed a new tool (SpliceScan II for predicting the effects of genetic variants on splicing and cis-regulatory elements. The novel Bayesian non-canonical 5'GC splice site (SS sensor used in our tool allows inference on non-canonical exons. Results Our tool performed favorably when compared with the existing methods in the context of genes linked to the Autism Spectrum Disorder (ASD. SpliceScan II was able to predict more aberrant splicing isoforms triggered by the mutations, as documented in DBASS5 and DBASS3 aberrant splicing databases, than other existing methods. Detrimental effects behind some of the polymorphic variations previously associated with Alzheimer's and breast cancer could be explained by changes in predicted splicing patterns. Conclusions We have developed SpliceScan II, an effective and sensitive tool for predicting the detrimental effects of genomic variants on splicing leading to Mendelian and complex hereditary disorders. The method could potentially be used to screen resequenced patient DNA to identify de novo mutations and polymorphic variants that could contribute to a genetic disorder.

  19. Taxonomic richness and abundance of cryptic peracarid crustaceans in the Puerto Morelos Reef National Park, Mexico

    Directory of Open Access Journals (Sweden)

    Luz Veronica Monroy-Velázquez

    2017-06-01

    Full Text Available Background and Aims Cryptic peracarids are an important component of the coral reef fauna in terms of diversity and abundance, yet they have been poorly studied. The aim of this study was to evaluate the taxonomic richness and abundance of cryptic peracarids in coral rubble in the Puerto Morelos Reef National Park, Mexico (PMRNP, and their relationship with depth. Methods Three reef sites were selected: (1 Bonanza, (2 Bocana, and (3 Jardines. At each site six kilograms of coral rubble were collected over four sampling periods at three depths: 3 m (back-reef, 6–8 m (fore-reef, and 10–12 m (fore-reef. Results A total of 8,887 peracarid crustaceans belonging to 200 taxa distributed over five orders and 63 families was obtained; 70% of the taxa were identified to species and 25% to genus level. Fifty species of those collected represent new records for the Mexican Caribbean Sea. Isopoda was the most speciose order while Tanaidacea was the most abundant. Discussion Cryptic peracarid taxonomic richness and abundance were related to depth with higher values of both parameters being found in the shallow (3 m back-reef, possibly due to a higher reef development and a greater accumulation of coral rubble produced during hurricanes. Peracarid data obtained in the present study can be used as a baseline for future monitoring programs in the PMRNP.

  20. Taxonomic richness and abundance of cryptic peracarid crustaceans in the Puerto Morelos Reef National Park, Mexico.

    Science.gov (United States)

    Monroy-Velázquez, Luz Veronica; Rodríguez-Martínez, Rosa Elisa; Alvarez, Fernando

    2017-01-01

    Cryptic peracarids are an important component of the coral reef fauna in terms of diversity and abundance, yet they have been poorly studied. The aim of this study was to evaluate the taxonomic richness and abundance of cryptic peracarids in coral rubble in the Puerto Morelos Reef National Park, Mexico (PMRNP), and their relationship with depth. Three reef sites were selected: (1) Bonanza, (2) Bocana, and (3) Jardines. At each site six kilograms of coral rubble were collected over four sampling periods at three depths: 3 m (back-reef), 6-8 m (fore-reef), and 10-12 m (fore-reef). A total of 8,887 peracarid crustaceans belonging to 200 taxa distributed over five orders and 63 families was obtained; 70% of the taxa were identified to species and 25% to genus level. Fifty species of those collected represent new records for the Mexican Caribbean Sea. Isopoda was the most speciose order while Tanaidacea was the most abundant. Cryptic peracarid taxonomic richness and abundance were related to depth with higher values of both parameters being found in the shallow (3 m) back-reef, possibly due to a higher reef development and a greater accumulation of coral rubble produced during hurricanes. Peracarid data obtained in the present study can be used as a baseline for future monitoring programs in the PMRNP.

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

    Science.gov (United States)

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

    2017-04-05

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

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-11-07

    In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.

  4. Targeting RNA splicing for disease therapy.

    Science.gov (United States)

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

    2013-01-01

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

  5. Cryptic speciation in a model invertebrate chordate.

    Science.gov (United States)

    Caputi, Luigi; Andreakis, Nikos; Mastrototaro, Francesco; Cirino, Paola; Vassillo, Mauro; Sordino, Paolo

    2007-05-29

    We applied independent species concepts to clarify the phylogeographic structure of the ascidian Ciona intestinalis, a powerful model system in chordate biology and for comparative genomic studies. Intensive research with this marine invertebrate is based on the assumption that natural populations globally belong to a single species. Therefore, understanding the true taxonomic classification may have implications for experimental design and data management. Phylogenies inferred from mitochondrial and nuclear DNA markers accredit the existence of two cryptic species: C. intestinalis sp. A, genetically homogeneous, distributed in the Mediterranean, northeast Atlantic, and Pacific, and C. intestinalis sp. B, geographically structured and encountered in the North Atlantic. Species-level divergence is further entailed by cross-breeding estimates. C. intestinalis A and B from allopatric populations cross-fertilize, but hybrids remain infertile because of defective gametogenesis. Although anatomy illustrates an overall interspecific similarity lacking in diagnostic features, we provide consistent tools for in-field and in-laboratory species discrimination. Finding of two cryptic taxa in C. intestinalis raises interest in a new tunicate genome as a gateway to studies in speciation and ecological adaptation of chordates.

  6. Familial cryptic translocation in Angelman syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Weyerts, L.K.; Wiley, J.E.; Loud, K.M. [ECU School of Medicine, Greenville, NC (United States)] [and others

    1994-09-01

    The majority of patients with Angelman syndrome have been shown to have a cytogenetic or molecular deletion on the maternally derived chromosome 15. We report on a case of Angelman syndrome in which this deletion occurs as an unbalanced cryptic translocation involving chromosomes 14 and 15. The proband was diagnosed clinically as having Angelman syndrome. Multiple cytogenetic studies were done without detecting any deletion. When DNA probes (Oncor) specific for the Prader Willi/Angelman locus became available, the patient was restudied and found to be deleted for {open_quotes}region A{close_quotes} (D15S11) but not for {open_quotes}region B{close_quotes} (GABRB3). No other abnormality was detected. The proband`s mother was then studied. The chromosome 15 marker probe and D15S11 were detected on different chromosomes. Using alpha-satellite probes, a cryptic 14;15 translocation was uncovered. This balanced translocation was also found to be carried by the sister of the proband. This case, along with a case presented at the 1993 ASHG meeting, illustrates the need for using acrocentric probes when studying Angelman syndrome patients. The proband was studied using additional probes specific for this region and found to be deleted for SNRPN but not for D15S10. The breakpoint of the translocation in this patient delineates the smallest deletion of the Angelman syndrome region reported to date and therefore may represent the specific gene involved.

  7. A Combinatorial Code for Splicing Silencing: UAGG and GGGG Motifs: e158

    National Research Council Canada - National Science Library

    Kyoungha Han; Gene Yeo; Ping An; Christopher B Burge; Paula J Grabowski

    2005-01-01

    .... Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5'-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19...

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

    Science.gov (United States)

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

    2010-04-01

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

  9. Alternative splicing regulates targeting of malate dehydrogenase in Yarrowia lipolytica.

    Science.gov (United States)

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

    2012-06-01

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

  10. Splign: algorithms for computing spliced alignments with identification of paralogs

    Directory of Open Access Journals (Sweden)

    Tatusova Tatiana

    2008-05-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

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

    2011-07-01

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

  12. Methods for Characterization of Alternative RNA Splicing

    Science.gov (United States)

    Harvey, Samuel E.; Cheng, Chonghui

    2016-01-01

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

  13. A combinatorial code for splicing silencing: UAGG and GGGG motifs.

    Directory of Open Access Journals (Sweden)

    Kyoungha Han

    2005-05-01

    Full Text Available Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5'-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19 of the glutamate NMDA R1 receptor (GRIN1 transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes.

  14. A combinatorial code for splicing silencing: UAGG and GGGG motifs.

    Science.gov (United States)

    Han, Kyoungha; Yeo, Gene; An, Ping; Burge, Christopher B; Grabowski, Paula J

    2005-05-01

    Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5'-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes.

  15. In vitro splicing of erythropoietin by the Mycobacterium tuberculosis RecA intein without substituting amino acids at the splice junctions.

    Science.gov (United States)

    Gangopadhyay, Jaya Pal; Jiang, Shu-qin; van Berkel, Patrick; Paulus, Henry

    2003-01-20

    Protein splicing is a self-catalyzed process involving the excision of an intervening polypeptide sequence, the intein, and joining of the flanking polypeptide sequences, the extein, by a peptide bond. We have studied the in vitro splicing of erythropoietin (EPO) using a truncated form of the Mycobacterium tuberculosis RecA mini-intein in which the homing endonuclease domain was replaced with a hexahistidine sequence (His-tag). The intein was inserted adjacent to cysteine residues to assure that the spliced product had the natural amino acid sequence. When expressed in Escherichia coli, intein-containing EPO was found entirely as inclusion bodies but could be refolded in soluble form in the presence of 0.5 M arginine. Protein splicing of the refolded protein could be induced with a reducing agent such as DTT or tris(2-carboxyethyl)phosphine and led to the formation of EPO and mini-intein along with some cleavage products. Protein splicing mediated by the RecA intein requires the presence of a cysteine residue adjacent to the intein insertion site. We compared the efficiencies of protein splicing adjacent to three of the four cysteine residues of EPO (Cys29, Cys33 and Cys161) and found that insertion of intein adjacent to Cys29 allowed far more efficient protein splicing than insertion adjacent to Cys33 or Cys161. For ease of purification, our experiments involved a His-tagged EPO fusion protein and a His-tagged intein and the spliced products (25 kDa EPO and 24 kDa mini-intein) were identified by Western blotting using anti-EPO and anti-His-tag antibodies and by mass spectroscopy. The optimal splicing yield at Cys29 (40%) occurred at pH 7.0 after refolding at 4 degrees C and splicing for 18 h at 25 degrees C in the presence of 1 mM DTT.

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

    Directory of Open Access Journals (Sweden)

    María E. Teresa-Rodrigo

    2014-06-01

    Full Text Available Cornelia de Lange syndrome (CdLS is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21 or functionally associated factors (NIPBL, HDAC8 of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ΔE10, ΔE12, ΔE33,34, and B’. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame.

  17. Cryptic species in putative ancient asexual darwinulids (Crustacea, Ostracoda.

    Directory of Open Access Journals (Sweden)

    Isa Schön

    Full Text Available BACKGROUND: Fully asexually reproducing taxa lack outcrossing. Hence, the classic Biological Species Concept cannot be applied. METHODOLOGY/PRINCIPAL FINDINGS: We used DNA sequences from the mitochondrial COI gene and the nuclear ITS2 region to check species boundaries according to the evolutionary genetic (EG species concept in five morphospecies in the putative ancient asexual ostracod genera, Penthesilenula and Darwinula, from different continents. We applied two methods for detecting cryptic species, namely the K/θ method and the General Mixed Yule Coalescent model (GMYC. We could confirm the existence of species in all five darwinulid morphospecies and additional cryptic diversity in three morphospecies, namely in Penthesilenula brasiliensis, Darwinula stevensoni and in P. aotearoa. The number of cryptic species within one morphospecies varied between seven (P. brasiliensis, five to six (D. stevensoni and two (P. aotearoa, respectively, depending on the method used. Cryptic species mainly followed continental distributions. We also found evidence for coexistence at the local scale for Brazilian cryptic species of P. brasiliensis and P. aotearoa. Our ITS2 data confirmed that species exist in darwinulids but detected far less EG species, namely two to three cryptic species in P. brasiliensis and no cryptic species at all in the other darwinulid morphospecies. CONCLUSIONS/SIGNIFICANCE: Our results clearly demonstrate that both species and cryptic diversity can be recognized in putative ancient asexual ostracods using the EG species concept, and that COI data are more suitable than ITS2 for this purpose. The discovery of up to eight cryptic species within a single morphospecies will significantly increase estimates of biodiversity in this asexual ostracod group. Which factors, other than long-term geographic isolation, are important for speciation processes in these ancient asexuals remains to be investigated.

  18. SOAPsplice: genome-wide ab initio detection of splice junctions from RNA-Seq data

    Directory of Open Access Journals (Sweden)

    Songbo eHuang

    2011-07-01

    Full Text Available RNA-Seq, a method using next generation sequencing technologies to sequence the transcriptome, facilitates genome-wide analysis of splice junction sites. In this paper, we introduce SOAPsplice, a robust tool to detect splice junctions using RNA-Seq data without using any information of known splice junctions. SOAPsplice uses a novel two-step approach consisting of first identifying as many reasonable splice junction candidates as possible, and then, filtering the false positives with two effective filtering strategies. In both simulated and real datasets, SOAPsplice is able to detect many reliable splice junctions with low false positive rate. The improvement gained by SOAPsplice, when compared to other existing tools, becomes more obvious when the depth of sequencing is low. SOAPsplice is freely available at http://soap.genomics.org.cn/soapsplice.html.

  19. Comprehensive analysis of alternative splicing in rice and comparative analyses with Arabidopsis

    Directory of Open Access Journals (Sweden)

    Mount Stephen M

    2006-12-01

    Full Text Available Abstract Background Recently, genomic sequencing efforts were finished for Oryza sativa (cultivated rice and Arabidopsis thaliana (Arabidopsis. Additionally, these two plant species have extensive cDNA and expressed sequence tag (EST libraries. We employed the Program to Assemble Spliced Alignments (PASA to identify and analyze alternatively spliced isoforms in both species. Results A comprehensive analysis of alternative splicing was performed in rice that started with >1.1 million publicly available spliced ESTs and over 30,000 full length cDNAs in conjunction with the newly enhanced PASA software. A parallel analysis was performed with Arabidopsis to compare and ascertain potential differences between monocots and dicots. Alternative splicing is a widespread phenomenon (observed in greater than 30% of the loci with transcript support and we have described nine alternative splicing variations. While alternative splicing has the potential to create many RNA isoforms from a single locus, the majority of loci generate only two or three isoforms and transcript support indicates that these isoforms are generally not rare events. For the alternate donor (AD and acceptor (AA classes, the distance between the splice sites for the majority of events was found to be less than 50 basepairs (bp. In both species, the most frequent distance between AA is 3 bp, consistent with reports in mammalian systems. Conversely, the most frequent distance between AD is 4 bp in both plant species, as previously observed in mouse. Most alternative splicing variations are localized to the protein coding sequence and are predicted to significantly alter the coding sequence. Conclusion Alternative splicing is widespread in both rice and Arabidopsis and these species share many common features. Interestingly, alternative splicing may play a role beyond creating novel combinations of transcripts that expand the proteome. Many isoforms will presumably have negative

  20. Experimental demonstration of possible cryptic female choice on male tsetse fly genitalia.

    Science.gov (United States)

    Briceño, R D; Eberhard, W G

    2009-11-01

    A possible explanation for one of the most general trends in animal evolution - rapid divergent evolution of animal genitalia - is that male genitalia are used as courtship devices that influence cryptic female choice. But experimental demonstrations of stimulatory effects of male genitalia on female reproductive processes have generally been lacking. Previous studies of female reproductive physiology in the tsetse fly Glossina morsitans suggested that stimulation during copulation triggers ovulation and resistance to remating. In this study we altered the form of two male genital structures that squeeze the female's abdomen rhythmically in G. morsitans centralis and induced, as predicted, cryptic female choice against the male: sperm storage decreased, while female remating increased. Further experiments in which we altered the female sensory abilities at the site contacted by these male structures during copulation, and severely altered or eliminated the stimuli the male received from this portion of his genitalia, suggested that the effects of genital alteration on sperm storage were due to changes in tactile stimuli received by the female, rather than altered male behavior. These data support the hypothesis that sexual selection by cryptic female choice has been responsible for the rapid divergent evolution of male genitalia in Glossina; limitations of this support are discussed. It appears that a complex combination of stimuli trigger female ovulation, sperm storage, and remating, and different stimuli affect different processes in G. morsitans, and that the same processes are controlled differently in G. pallidipes. This puzzling diversity in female triggering mechanisms may be due to the action of sexual selection.

  1. Evidence of female cryptic choice in crayfish.

    Science.gov (United States)

    Aquiloni, Laura; Gherardi, Francesca

    2008-04-23

    To test whether male body size affects female reproductive investment in the polygamous crayfish Procambarus clarkii, we described mating behaviour of virgin females paired with either small or large males, and analysed the number, size and weight of both eggs and juveniles sired by either types of male. Along with confirming the overt selection by females of larger mates, we found that the size and weight of both the eggs and the juveniles were higher when sired by larger fathers. This suggests that P. clarkii females exert a form of cryptic choice for large males, seemingly adjusting the quantity of egg deutoplasm in function of the mate body size. The question of why females spend time and energy to brood low-fitness offspring is finally raised.

  2. Drug design from the cryptic inhibitor envelope.

    Science.gov (United States)

    Lee, Chul-Jin; Liang, Xiaofei; Wu, Qinglin; Najeeb, Javaria; Zhao, Jinshi; Gopalaswamy, Ramesh; Titecat, Marie; Sebbane, Florent; Lemaitre, Nadine; Toone, Eric J; Zhou, Pei

    2016-02-25

    Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC--an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target--access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics.

  3. Drug design from the cryptic inhibitor envelope

    Science.gov (United States)

    Lee, Chul-Jin; Liang, Xiaofei; Wu, Qinglin; Najeeb, Javaria; Zhao, Jinshi; Gopalaswamy, Ramesh; Titecat, Marie; Sebbane, Florent; Lemaitre, Nadine; Toone, Eric J.; Zhou, Pei

    2016-01-01

    Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC—an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target—access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics. PMID:26912110

  4. Unusual intron conservation near tissue-regulated exons found by splicing microarrays.

    Directory of Open Access Journals (Sweden)

    Charles W Sugnet

    2006-01-01

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

  5. HP1 Is Involved in Regulating the Global Impact of DNA Methylation on Alternative Splicing

    Directory of Open Access Journals (Sweden)

    Ahuvi Yearim

    2015-02-01

    Full Text Available The global impact of DNA methylation on alternative splicing is largely unknown. Using a genome-wide approach in wild-type and methylation-deficient embryonic stem cells, we found that DNA methylation can either enhance or silence exon recognition and affects the splicing of more than 20% of alternative exons. These exons are characterized by distinct genetic and epigenetic signatures. Alternative splicing regulation of a subset of these exons can be explained by heterochromatin protein 1 (HP1, which silences or enhances exon recognition in a position-dependent manner. We constructed an experimental system using site-specific targeting of a methylated/unmethylated gene and demonstrate a direct causal relationship between DNA methylation and alternative splicing. HP1 regulates this gene’s alternative splicing in a methylation-dependent manner by recruiting splicing factors to its methylated form. Our results demonstrate DNA methylation’s significant global influence on mRNA splicing and identify a specific mechanism of splicing regulation mediated by HP1.

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

    Science.gov (United States)

    Elina, Helen; Brown, Gregory G

    2010-01-01

    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.

  7. Splice testing for LHC quadrupole magnets

    CERN Document Server

    Barzi, E; Fehér, S; Kashikhin, V V; Kerby, J S; Lamm, M J; Orris, D; Ray, G; Tartaglia, M; Zlobin, A V

    2003-01-01

    Electrical splices between NbTi Rutherford type cables need to be made for the LHC IR inner triplet quadrupoles. Splices between magnets as well as internal to the magnets are necessary. Various splice configurations, solders, and fluxes have been considered. Testing of these splices at cryogenic temperatures and at various currents has been completed. The results were satisfactory; Fermilab is capable of making excellent low resistance (<1n Omega ) solder joints for the LHC project. (4 refs).

  8. Stochastic noise in splicing machinery

    OpenAIRE

    Melamud, Eugene; Moult, John

    2009-01-01

    The number of known alternative human isoforms has been increasing steadily with the amount of available transcription data. To date, over 100 000 isoforms have been detected in EST libraries, and at least 75% of human genes have at least one alternative isoform. In this paper, we propose that most alternative splicing events are the result of noise in the splicing process. We show that the number of isoforms and their abundance can be predicted by a simple stochastic noise model that takes i...

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

    Science.gov (United States)

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

    2013-01-01

    For most of our 25,000 genes, the removal of introns by pre-messenger RNA (pre-mRNA) splicing represents an essential step toward the production of functional messenger RNAs (mRNAs). Alternative splicing of a single pre-mRNA results in the production of different mRNAs. Although complex organisms use alternative splicing to expand protein function and phenotypic diversity, patterns of alternative splicing are often altered in cancer cells. Alternative splicing contributes to tumorigenesis by producing splice isoforms that can stimulate cell proliferation and cell migration or induce resistance to apoptosis and anticancer agents. Cancer-specific changes in splicing profiles can occur through mutations that are affecting splice sites and splicing control elements, and also by alterations in the expression of proteins that control splicing decisions. Recent progress in global approaches that interrogate splicing diversity should help to obtain specific splicing signatures for cancer types. The development of innovative approaches for annotating and reprogramming splicing events will more fully establish the essential contribution of alternative splicing to the biology of cancer and will hopefully provide novel targets and anticancer strategies. Metazoan genes are usually made up of several exons interrupted by introns. The introns are removed from the pre-mRNA by RNA splicing. In conjunction with other maturation steps, such as capping and polyadenylation, the spliced mRNA is then transported to the cytoplasm to be translated into a functional protein. The basic mechanism of splicing requires accurate recognition of each extremity of each intron by the spliceosome. Introns are identified by the binding of U1 snRNP to the 5' splice site and the U2AF65/U2AF35 complex to the 3' splice site. Following these interactions, other proteins and snRNPs are recruited to generate the complete spliceosomal complex needed to excise the intron. While many introns are constitutively

  10. EHLERS-DANLOS SYNDROME TYPE-IV - PHENOTYPIC CONSEQUENCES OF A SPLICING MUTATION IN ONE COL3A1 ALLELE

    NARCIS (Netherlands)

    SILLENCE, DO; CHIODO, AA; CAMPBELL, PE; COLE, WG

    1991-01-01

    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(II

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

    Science.gov (United States)

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

    2013-01-01

    The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of organisms. These factors affect splicing through both positive and negative controls of splice site recognition by pre-spliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferase-based splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2-dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68, and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2-dependent intron inclusion. PMID:23880637

  12. Fine-scale variation and genetic determinants of alternative splicing across individuals.

    Directory of Open Access Journals (Sweden)

    Jasmin Coulombe-Huntington

    2009-12-01

    Full Text Available 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

  13. PTBP1-dependent regulation of USP5 alternative RNA splicing plays a role in glioblastoma tumorigenesis.

    Science.gov (United States)

    Izaguirre, Daisy I; Zhu, Wen; Hai, Tao; Cheung, Hannah C; Krahe, Ralf; Cote, Gilbert J

    2012-11-01

    Aberrant RNA splicing is thought to play a key role in tumorigenesis. The assessment of its specific contributions is limited by the complexity of information derived from genome-wide array-based approaches. We describe how performing splicing factor-specific comparisons using both tumor and cell line data sets may more readily identify physiologically relevant tumor-specific splicing events. Affymetrix exon array data derived from glioblastoma (GBM) tumor samples with defined polypyrimidine tract-binding protein 1 (PTBP1) levels were compared with data from U251 GBM cells with and without PTBP1 knockdown. This comparison yielded overlapping gene sets that comprised only a minor fraction of each data set. The identification of a novel GBM-specific splicing event involving the USP5 gene led us to further examine its role in tumorigenesis. In GBM, USP5 generates a shorter isoform 2 through recognition of a 5' splice site within exon 15. Production of the USP5 isoform 2 was strongly correlated with PTBP1 expression in GBM tumor samples and cell lines. Splicing regulation was consistent with the presence of an intronic PTBP1 binding site and could be modulated through antisense targeting of the isoform 2 splice site to force expression of isoform 1 in GBM cells. The forced expression of USP5 isoform 1 in two GBM cell lines inhibited cell growth and migration, implying an important role for USP5 splicing in gliomagenesis. These results support a role for aberrant RNA splicing in tumorigenesis and suggest that changes in relatively few genes may be sufficient to drive the process.

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

    Science.gov (United States)

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

    2010-09-01

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

  15. Modification of Alternative Splicing of Bcl-x Pre-mRNA in Bladder Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    ZHU Zhaohui; XING Shi'an; CHENG Ping; ZENG Fuqing; LU Gongcheng

    2006-01-01

    To modify the splicing pattern of Bcl-x and compare the effect of this approach with that of the antisense gene therapy in BIU-87 cell line of bladder cancer, by using 5'-Bcl-x AS to target downstream alternative 5'-Bcl-x splice site to shift splicing from Bcl-xL to Bcl-xS and 3'-Bcl-x AS antisense to the 3'-splice site of exon Ⅲ in Bcl-x pre- mRNA to down regulation of Bcl-xL expression,the inhibitory effects on cancer cells by modification of alternative splicing and antisense gene therapy were observed and compared by microscopy, MTT Assay, RT-PCR, FACS, Westhern bloting and clone formation. The growth of cells BIU-87 was inhibited in a dose- and time-dependent manner. Its inhibitory effect began 12 h after the exposure, reaching a maximum value after 72h. The number of cells decreased in S phase and the number increased in G1 phase. The ability to form foci was reduced and the antisense gene therapy was approximately half as efficient as modification of alternative splicing in inducing apoptosis. It is concluded that modification of splicing pattern of Bcl-x pre-mRNA in bladder cancer cell BIU-87 is better than antisense gene therapy in terms of tumor inhibition.

  16. Stochastic noise in splicing machinery.

    Science.gov (United States)

    Melamud, Eugene; Moult, John

    2009-08-01

    The number of known alternative human isoforms has been increasing steadily with the amount of available transcription data. To date, over 100 000 isoforms have been detected in EST libraries, and at least 75% of human genes have at least one alternative isoform. In this paper, we propose that most alternative splicing events are the result of noise in the splicing process. We show that the number of isoforms and their abundance can be predicted by a simple stochastic noise model that takes into account two factors: the number of introns in a gene and the expression level of a gene. The results strongly support the hypothesis that most alternative splicing is a consequence of stochastic noise in the splicing machinery, and has no functional significance. The results are also consistent with error rates tuned to ensure that an adequate level of functional product is produced and to reduce the toxic effect of accumulation of misfolding proteins. Based on simulation of sampling of virtual cDNA libraries, we estimate that error rates range from 1 to 10% depending on the number of introns and the expression level of a gene.

  17. Tdp-43 cryptic exons are highly variable between cell types.

    Science.gov (United States)

    Jeong, Yun Ha; Ling, Jonathan P; Lin, Sophie Z; Donde, Aneesh N; Braunstein, Kerstin E; Majounie, Elisa; Traynor, Bryan J; LaClair, Katherine D; Lloyd, Thomas E; Wong, Philip C

    2017-02-02

    TDP-43 proteinopathy is a prominent pathological feature that occurs in a number of human diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and inclusion body myositis (IBM). Our recent finding that TDP-43 represses nonconserved cryptic exons led us to ask whether cell type-specific cryptic exons could exist to impact unique molecular pathways in brain or muscle. In the present work, we investigated TDP-43's function in various mouse tissues to model disease pathogenesis. We generated mice to conditionally delete TDP-43 in excitatory neurons or skeletal myocytes and identified the cell type-specific cryptic exons associated with TDP-43 loss of function. Comparative analysis of nonconserved cryptic exons in various mouse cell types revealed that only some cryptic exons were common amongst stem cells, neurons, and myocytes; the majority of these nonconserved cryptic exons were cell type-specific. Our results suggest that in human disease, TDP-43 loss of function may impair cell type-specific pathways.

  18. Negative and positive mRNA splicing elements act competitively to regulate human immunodeficiency virus type 1 vif gene expression.

    Science.gov (United States)

    Exline, C M; Feng, Z; Stoltzfus, C M

    2008-04-01

    Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3' splice sites (3'ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3'ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5' splice site (5'ss) downstream of exon 2 (5'ss D2). Here we show that the mutations within 5'ss D2 that are predicted to lower or increase the affinity of the 5'ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5'ss D2 was not necessary for the effect of 5'ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5'ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5'-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5'ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication.

  19. Alcoholism and alternative splicing of candidate genes.

    Science.gov (United States)

    Sasabe, Toshikazu; Ishiura, Shoichi

    2010-04-01

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

  20. Evolutionary conservation of alternative splicing in chicken

    Science.gov (United States)

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

    2013-01-01

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

  1. COMMUNICATION: Alternative splicing and genomic stability

    Science.gov (United States)

    Cahill, Kevin

    2004-06-01

    Alternative splicing allows an organism to make different proteins in different cells at different times, all from the same gene. In a cell that uses alternative splicing, the total length of all the exons is much shorter than in a cell that encodes the same set of proteins without alternative splicing. This economical use of exons makes genes more stable during reproduction and development because a genome with a shorter exon length is more resistant to harmful mutations. Genomic stability may be the reason why higher vertebrates splice alternatively. For a broad class of alternatively spliced genes, a formula is given for the increase in their stability.

  2. Functional consequences of splicing of the antisense transcript COOLAIR on FLC transcription

    DEFF Research Database (Denmark)

    Marquardt, Sebastian; Raitskin, Oleg; Wu, Zhe

    2014-01-01

    perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor...... site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms.......Antisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just...

  3. Salt-Dependent Conditional Protein Splicing of an Intein from Halobacterium salinarum.

    Science.gov (United States)

    Reitter, Julie N; Cousin, Christopher E; Nicastri, Michael C; Jaramillo, Mario V; Mills, Kenneth V

    2016-03-01

    An intein from Halobacterium salinarum can be isolated as an unspliced precursor protein with exogenous exteins after Escherichia coli overexpression. The intein promotes protein splicing and uncoupled N-terminal cleavage in vitro, conditional on incubation with NaCl or KCl at concentrations of >1.5 M. The protein splicing reaction also is conditional on reduction of a disulfide bond between two active site cysteines. Conditional protein splicing under these relatively mild conditions may lead to advances in intein-based biotechnology applications and hints at the possibility that this H. salinarum intein could serve as a switch to control extein activity under physiologically relevant conditions.

  4. Evidence that U2/U6 helix I promotes both catalytic steps of pre-mRNA splicing and rearranges in between these steps.

    Science.gov (United States)

    Mefford, Melissa A; Staley, Jonathan P

    2009-07-01

    During pre-mRNA splicing, the spliceosome must configure the substrate, catalyze 5' splice site cleavage, reposition the substrate, and catalyze exon ligation. The highly conserved U2/U6 helix I, which adjoins sequences that define the reactive sites, has been proposed to configure the substrate for 5' splice site cleavage and promote catalysis. However, a role for this helix at either catalytic step has not been tested rigorously and previous observations question its role at the catalytic steps. Through a comprehensive molecular genetic study of U2/U6 helix I, we found that weakening U2/U6 helix I, but not mutually exclusive structures, compromised splicing of a substrate limited at the catalytic step of 5' splice site cleavage, providing the first compelling evidence that this helix indeed configures the substrate during 5' splice site cleavage. Further, mutations that we proved weaken only U2/U6 helix I suppressed a mutation in PRP16, a DEAH-box ATPase required after 5' splice site cleavage, providing persuasive evidence that helix I is destabilized by Prp16p and suggesting that this structure is unwound between the catalytic steps. Lastly, weakening U2/U6 helix I also compromised splicing of a substrate limited at the catalytic step of exon ligation, providing evidence that U2/U6 helix I reforms and functions during exon ligation. Thus, our data provide evidence for a fundamental and apparently dynamic role for U2/U6 helix I during the catalytic stages of splicing.

  5. Titin Diversity—Alternative Splicing Gone Wild

    Directory of Open Access Journals (Sweden)

    Wei Guo

    2010-01-01

    Full Text Available Titin is an extremely large protein found in highest concentrations in heart and skeletal muscle. The single mammalian gene is expressed in multiple isoforms as a result of alternative splicing. Although titin isoform expression is controlled developmentally and in a tissue specific manner, the vast number of potential splicing pathways far exceeds those described in any other alternatively spliced gene. Over 1 million human splice pathways for a single individual can be potentially derived from the PEVK region alone. A new splicing pattern for the human cardiac N2BA isoform type has been found in which the PEVK region includes only the N2B type exons. The alterations in splicing and titin isoform expression in human heart disease provide impetus for future detailed study of the splicing mechanisms for this giant protein.

  6. Alternative pre-mRNA splicing in Drosophila spliceosomal assembly factor RNP-4F during development.

    Science.gov (United States)

    Fetherson, Rebecca A; Strock, Stephen B; White, Kristen N; Vaughn, Jack C

    2006-04-26

    The 5'- and 3'-UTR regions in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation. Here we report the results of a systematic study of alternative splicing in rnp-4f, which encodes a Drosophila spliceosomal assembly factor. We show that most of the nine introns are constitutively spliced, but several patterns of alternative splicing are observed in two pre-mRNA regions including the 5'-UTR. Intron V is shown to be of recent evolutionary origin and is infrequently spliced, resulting in generation of an in-frame stop codon and a predicted truncated protein lacking a nuclear localization signal, so that alternative splicing regulates its subcellular localization. Intron 0, located in the 5'-UTR, is subject to three different splicing decisions in D. melanogaster. Northern analysis of poly(A+) mRNAs reveals two differently sized rnp-4f mRNA isoforms in this species. A switch in relative isoform abundance occurs during mid-embryo stages, when the larger isoform becomes more abundant. This isoform is shown to represent intron 0 unspliced mRNA, whereas the smaller transcript represents the product of alternative splicing. Comparative genomic analysis predicts that intron 0 is present in diverse Drosophila species. Intron 0 splicing results in loss of an evolutionarily conserved stem-loop constituting a potential cis-regulatory element at the 3'-splice site. A model is proposed for the role of this element both in 5'-UTR alternative splicing decisions and in RNP-4F translational modulation. Preliminary evidences in support of our model are discussed.

  7. Identification of novel splice variants of Adhesion G protein-coupled receptors.

    Science.gov (United States)

    Bjarnadóttir, Thóra K; Geirardsdóttir, Kristín; Ingemansson, Malena; Mirza, Majd A I; Fredriksson, Robert; Schiöth, Helgi B

    2007-01-31

    Alternative splicing is an important mechanism to generate proteome diversity in higher eukaryotic organisms. We searched for splice variants of the human Adhesion family of G protein-coupled receptors (GPCRs) using mRNA sequences and expressed sequence tags. The results presented here describe 53 human splice variants among the 33 Adhesion GPCRs. Many of these variants appear to be coding for "functional" proteins (29) while the others are seemingly "non-functional" (24). Novel functional splice variants were found for: CD97, CELR3, EMR2, EMR3, GPR56, GPR110, GPR112-GPR114, GPR116, GPR123-GPR126, GPR133, HE6, and LEC1-LEC3. Splice variants for GPR116, GPR125, GPR126, and HE6 were found conserved in other species. Several of the functional splice variants lack one or more of the functional domains that are found in the N-termini of these receptors. These functional domains are likely to affect ligand binding or interaction with other proteins and these novel splice variants may have important roles for the specificity of interactions between these receptors and extracellular molecules. Another type of splice variants found here lacks a GPCR proteolytic site (GPS). The GPS domain has been shown to be essential for the proteolytic cleavage of the receptors N-termini and for cellular surface expression. We suggest that these alternative splice variants may be crucial for the function of the receptors while the seemingly non-functional splice variants may be a part of a regulative mechanism.

  8. Position-dependent repression and promotion of DQB1 intron 3 splicing by GGGG motifs.

    Science.gov (United States)

    Královicová, Jana; Vorechovsky, Igor

    2006-02-15

    Alternative splicing of HLA-DQB1 exon 4 is allele-dependent and results in variable expression of soluble DQbeta. We have recently shown that differential inclusion of this exon in mature transcripts is largely due to intron 3 variants in the branch point sequence (BPS) and polypyrimidine tract. To identify additional regulatory cis-elements that contribute to haplotype-specific splicing of DQB1, we systematically examined the effect of guanosine (G) repeats on intron 3 removal. We found that the GGG or GGGG repeats generally improved splicing of DQB1 intron 3, except for those that were adjacent to the 5' splice site where they had the opposite effect. The most prominent splicing enhancement was conferred by GGGG motifs arranged in tandem upstream of the BPS. Replacement of a G-rich segment just 5' of the BPS with a series of random sequences markedly repressed splicing, whereas substitutions of a segment further upstream that lacked the G-rich elements and had the same size did not result in comparable splicing inhibition. Systematic mutagenesis of both suprabranch guanosine quadruplets (G(4)) revealed a key role of central G residues in splicing enhancement, whereas cytosines in these positions had the most prominent repressive effects. Together, these results show a significant role of tandem G(4)NG(4) structures in splicing of both complete and truncated DQB1 intron 3, support position dependency of G repeats in splicing promotion and inhibition, and identify positively and negatively acting sequences that contribute to the haplotype-specific DQB1 expression.

  9. Alternative RNA splicing of KSHV ORF57 produces two different RNA isoforms.

    Science.gov (United States)

    Majerciak, Vladimir; Zheng, Zhi-Ming

    2016-01-15

    In lytically infected B cells Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 gene encodes two RNA isoforms by alternative splicing of its pre-mRNA, which contains a small, constitutive intron in its 5' half and a large, suboptimal intron in its 3's half. The RNA1 isoform encodes full-length ORF57 and is a major isoform derived from splicing of the constitutive small intron, but retaining the suboptimal large intron as the coding region. A small fraction (splicing to produce a smaller non-coding RNA2 due to lack of a translational termination codon. Both RNAs are cleaved and polyadenylated at the same cleavage site CS83636. The insertion of ORF57 RNA1 into a restriction cutting site in certain mammalian expression vectors activates splicing of the subopitmal intron and produces a truncated ORF57 protein.

  10. α-MSH regulates intergenic splicing of MC1R and TUBB3 in human melanocytes

    OpenAIRE

    Dalziel, Martin; Kolesnichenko, Marina; das Neves, Ricardo Pires; Iborra, Francisco; Goding, Colin; Furger, André

    2010-01-01

    Alternative splicing enables higher eukaryotes to increase their repertoire of proteins derived from a restricted number of genes. However, the possibility that functional diversity may also be augmented by splicing between adjacent genes has been largely neglected. Here, we show that the human melanocortin 1 receptor (MC1R) gene, a critical component of the facultative skin pigmentation system, has a highly complex and inefficient poly(A) site which is instrumental in allowing intergenic spl...

  11. Changes in exon–intron structure during vertebrate evolution affect the splicing pattern of exons

    Science.gov (United States)

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

    2012-01-01

    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

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

    Science.gov (United States)

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

    2016-01-01

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

  13. Insertion of part of an intron into the 5[prime] untranslated region of a Caenorhabditis elegans gene converts it into a trans-spliced gene

    Energy Technology Data Exchange (ETDEWEB)

    Conrad, R.; Thomas, J.; Spieth, J.; Blumenthal, T. (Indiana University, Bloomington (United States))

    1991-04-01

    In nematodes, the RNA products of some genes are trans-spliced to a 22-nucleotide spliced leader (SL), while the RNA products of other genes are not. In Caenorhabditis elegans, there are two SLs, Sl1 and SL2, donated by two distinct small nuclear ribonucleoprotein particles in a process functionally quite similar to nuclear intron removal. The authors demonstrate here that it is possible to convert a non-trans-spliced gene into a trans-spliced gene by placement of an intron missing only the 5[prime] splice site into the 5[prime] untranslated region. Stable transgenic strains were isolated expressing a gene in which 69 nucleotides of a vit-5 intron, including the 3[prime] splice site, were inserted into the 5[prime] untranslated region of a vit-2/vit-6 fusion gene. The RNA product of this gene was examined by primer extension and PCR amplification. Although the vit-2/vit-6 transgene product is not normally trans-spliced, the majority of transcripts from this altered gene were trans-spliced to SL1. They termed the region of a trans-spliced mRNA precursor between the 5[prime] end and the first 3[prime] splice site an 'outrun'. The results suggest that if a transcript begins with intronlike sequence followed by a 3[prime] splice site, this alone may constitute an outrun and be sufficient to demarcate a transcript as a trans-splice acceptor. These findings leave open the possibility that specific sequences are required to increase the efficiency of trans-splicing.

  14. Cryptic Genetic Variation in Evolutionary Developmental Genetics

    Directory of Open Access Journals (Sweden)

    Annalise B. Paaby

    2016-06-01

    Full Text Available Evolutionary developmental genetics has traditionally been conducted by two groups: Molecular evolutionists who emphasize divergence between species or higher taxa, and quantitative geneticists who study variation within species. Neither approach really comes to grips with the complexities of evolutionary transitions, particularly in light of the realization from genome-wide association studies that most complex traits fit an infinitesimal architecture, being influenced by thousands of loci. This paper discusses robustness, plasticity and lability, phenomena that we argue potentiate major evolutionary changes and provide a bridge between the conceptual treatments of macro- and micro-evolution. We offer cryptic genetic variation and conditional neutrality as mechanisms by which standing genetic variation can lead to developmental system drift and, sheltered within canalized processes, may facilitate developmental transitions and the evolution of novelty. Synthesis of the two dominant perspectives will require recognition that adaptation, divergence, drift and stability all depend on similar underlying quantitative genetic processes—processes that cannot be fully observed in continuously varying visible traits.

  15. Silencing of cryptic prophages in Corynebacterium glutamicum.

    Science.gov (United States)

    Pfeifer, Eugen; Hünnefeld, Max; Popa, Ovidiu; Polen, Tino; Kohlheyer, Dietrich; Baumgart, Meike; Frunzke, Julia

    2016-12-01

    DNA of viral origin represents a ubiquitous element of bacterial genomes. Its integration into host regulatory circuits is a pivotal driver of microbial evolution but requires the stringent regulation of phage gene activity. In this study, we describe the nucleoid-associated protein CgpS, which represents an essential protein functioning as a xenogeneic silencer in the Gram-positive Corynebacterium glutamicum CgpS is encoded by the cryptic prophage CGP3 of the C. glutamicum strain ATCC 13032 and was first identified by DNA affinity chromatography using an early phage promoter of CGP3. Genome-wide profiling of CgpS binding using chromatin affinity purification and sequencing (ChAP-Seq) revealed its association with AT-rich DNA elements, including the entire CGP3 prophage region (187 kbp), as well as several other elements acquired by horizontal gene transfer. Countersilencing of CgpS resulted in a significantly increased induction frequency of the CGP3 prophage. In contrast, a strain lacking the CGP3 prophage was not affected and displayed stable growth. In a bioinformatics approach, cgpS orthologs were identified primarily in actinobacterial genomes as well as several phage and prophage genomes. Sequence analysis of 618 orthologous proteins revealed a strong conservation of the secondary structure, supporting an ancient function of these xenogeneic silencers in phage-host interaction.

  16. Cryptic cuckoo eggs hide from competing cuckoos.

    Science.gov (United States)

    Gloag, Ros; Keller, Laurie-Anne; Langmore, Naomi E

    2014-10-07

    Interspecific arms races between cuckoos and their hosts have produced remarkable examples of mimicry, with parasite eggs evolving to match host egg appearance and so evade removal by hosts. Certain bronze-cuckoo species, however, lay eggs that are cryptic rather than mimetic. These eggs are coated in a low luminance pigment that camouflages them within the dark interiors of hosts' nests. We investigated whether cuckoo egg crypsis is likely to have arisen from the same coevolutionary processes known to favour egg mimicry. We added high and low luminance-painted eggs to the nests of large-billed gerygones (Gerygone magnirostris), a host of the little bronze-cuckoo (Chalcites minutillus). Gerygones rarely rejected either egg type, and did not reject natural cuckoo eggs. Cuckoos, by contrast, regularly removed an egg from clutches before laying their own and were five times more likely to remove a high luminance model than its low luminance counterpart. Given that we found one-third of all parasitized nests were exploited by multiple cuckoos, our results suggest that competition between cuckoos has been the key selective agent for egg crypsis. In such intraspecific arms races, crypsis may be favoured over mimicry because it can reduce the risk of egg removal to levels below chance.

  17. Cryptic Genetic Variation in Evolutionary Developmental Genetics.

    Science.gov (United States)

    Paaby, Annalise B; Gibson, Greg

    2016-06-13

    Evolutionary developmental genetics has traditionally been conducted by two groups: Molecular evolutionists who emphasize divergence between species or higher taxa, and quantitative geneticists who study variation within species. Neither approach really comes to grips with the complexities of evolutionary transitions, particularly in light of the realization from genome-wide association studies that most complex traits fit an infinitesimal architecture, being influenced by thousands of loci. This paper discusses robustness, plasticity and lability, phenomena that we argue potentiate major evolutionary changes and provide a bridge between the conceptual treatments of macro- and micro-evolution. We offer cryptic genetic variation and conditional neutrality as mechanisms by which standing genetic variation can lead to developmental system drift and, sheltered within canalized processes, may facilitate developmental transitions and the evolution of novelty. Synthesis of the two dominant perspectives will require recognition that adaptation, divergence, drift and stability all depend on similar underlying quantitative genetic processes-processes that cannot be fully observed in continuously varying visible traits.

  18. Spliced leader RNA trans-splicing discovered in copepods

    Science.gov (United States)

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A.; Sturm, Nancy R.; Liu, Guangxing; Zhang, Huan

    2015-12-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3‧-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods.

  19. Coral reef encruster communities and carbonate production in cryptic and exposed coral reef habitats along a gradient of terrestrial disturbance

    Science.gov (United States)

    Mallela, J.

    2007-12-01

    Encrusting calcareous organisms such as bryozoans, crustose coralline algae (CCA), foraminiferans, and serpulid worms are integral components of tropical framework-building reefs. They can contribute calcium carbonate to the reef framework, stabilise the substrate, and promote larval recruitment of other framework-building species (e.g. coral recruits). The percentage cover of encrusting organisms and their rates of carbonate production (g m-2 year-1) were assessed at four sites within a coastal embayment, along a gradient of riverine influence (high-low). As the orientation and type of substrate is thought to influence recruitment of encrusting organisms, organisms recruiting to both natural (the underside of platy corals) and experimental substrates were assessed. The effect of substrate exposure under different levels of riverine influence was assessed by orientating experimental substrates to mimic cryptic and exposed reef habitats (downwards-facing vs upwards-facing tiles) at each site. Cryptic experimental tiles supported similar encruster assemblages to those recruiting to the underneath (cryptic side) of platy corals, suggesting that tiles can be used as an experimental substrate to assess encruster recruitment in reef systems. Encruster cover, in particular CCA, and carbonate production was significantly higher at low-impact (clear water), high wave energy sites when compared to highly riverine impacted (turbid water), low wave energy sites. Cryptically orientated substrates supported a greater diversity of encrusting organisms, in particular serpulid worms and bryozoans. The inverse relationships observed between riverine inputs and encrusters (total encruster cover and carbonate production) have implications for both the current and future rates and styles of reefal framework production.

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

    Science.gov (United States)

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

    2016-01-01

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

  1. Automatic detection of exonic splicing enhancers (ESEs using SVMs

    Directory of Open Access Journals (Sweden)

    Suhai Sándor

    2008-09-01

    Full Text Available Abstract 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.

  2. Alternative Splicing in Plant Immunity

    Directory of Open Access Journals (Sweden)

    Shengming Yang

    2014-06-01

    Full Text Available Alternative splicing (AS occurs widely in plants and can provide the main source of transcriptome and proteome diversity in an organism. AS functions in a range of physiological processes, including plant disease resistance, but its biological roles and functional mechanisms remain poorly understood. Many plant disease resistance (R genes undergo AS, and several R genes require alternatively spliced transcripts to produce R proteins that can specifically recognize pathogen invasion. In the finely-tuned process of R protein activation, the truncated isoforms generated by AS may participate in plant disease resistance either by suppressing the negative regulation of initiation of immunity, or by directly engaging in effector-triggered signaling. Although emerging research has shown the functional significance of AS in plant biotic stress responses, many aspects of this topic remain to be understood. Several interesting issues surrounding the AS of R genes, especially regarding its functional roles and regulation, will require innovative techniques and additional research to unravel.

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

    Science.gov (United States)

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

    2017-01-01

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

  4. Alternative splicing in the differentiation of human embryonic stem cells into cardiac precursors.

    Directory of Open Access Journals (Sweden)

    Nathan Salomonis

    2009-11-01

    Full Text Available The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org, we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation.

  5. Unfolding the mystery of alternative splicing through a unique method of in vivo selection.

    Science.gov (United States)

    Singh, Ravindra N

    2007-05-01

    Alternative splicing of pre-messenger RNA (pre-mRNA) is a fundamental mechanism of gene regulation in higher eukaryotes. In addition to creating protein diversity, alternative splicing provides the safest mode of gene evolution. Of late, more and more forms of alternatively spliced transcripts (mRNAs) are being discovered for key genes. Some of the alternatively spliced transcripts are also associated with major human diseases. This has created a sense of urgency to find the methods by which regulation of alternative splicing of specific exons could be best understood. Here I review a powerful in vivo selection method that uses a combinatorial library of partially random sequences. Several advantages of this method include in vivo analysis of large sequences, identification of unique sequence motifs, determination of relative strength of splice sites and identification of long-distance interactions including role of RNA structures. This unique method could be applied to identify tissue-specific cis-elements. Similarly, the method is suitable to find cis-elements that become active in response to specific treatments of cells. Considering this unbiased method uses in vivo conditions, it has potential to identify critical regulatory elements as therapeutic targets for a growing number of splicing-associated diseases.

  6. Molecular identification and relative abundance of cryptic Lophodermium species in natural populations of Scots pine, Pinus sylvestris L.

    Science.gov (United States)

    Reignoux, Sabrina N A; Green, Sarah; Ennos, Richard A

    2014-01-01

    The multi-locus phylogenetic species recognition approach and population genetic analysis of Amplified Fragment Length Polymorphism (AFLP) markers were used to delineate Lophodermium taxa inhabiting needles of Scots pine (Pinus sylvestris) in native pinewoods within Scotland. These analyses revealed three major lineages corresponding to the morphological species Lophodermium seditiosum and Lophodermium conigenum, fruiting on broken branches, and Lophodermium pinastri, fruiting on naturally fallen needles. Within L. pinastri three well supported sister clades were found representing cryptic taxa designated L. pinastri I, L. pinastri II, and L. pinastri III. Significant differences in mean growth rate in culture were found among the cryptic taxa. Taxon-specific primers based on ITS sequences were designed and used to classify over 500 Lophodermium isolates, derived from fallen needles of P. sylvestris in three Scottish and one French pinewood site, into the three L. pinastri cryptic taxa. Highly significant differences in the relative abundance of the three taxa were found among the Scottish pinewood sites, and between the French and all of the Scottish sites.

  7. Characterization of a novel splicing variant in the RAPTOR gene

    Energy Technology Data Exchange (ETDEWEB)

    Sun Chang [Department of Human Genetics, University of Chicago, 920 E. 58th Street, Chicago, IL 60637 (United States)], E-mail: csun1@bsd.uchicago.edu; Southard, Catherine; Di Rienzo, Anna [Department of Human Genetics, University of Chicago, 920 E. 58th Street, Chicago, IL 60637 (United States)

    2009-03-09

    The mammalian target of rapamycin (mTOR) plays an essential role in the regulation of cell growth, proliferation and apoptosis. Raptor, the regulatory associated protein of mTOR, is an important member in this signaling pathway. In the present report, we identified and characterized a novel splicing variant of this gene, RAPTOR{sub v}2, in which exons 14-17, 474 bp in total, are omitted from the mRNA. This deletion does not change the open reading frame, but causes a nearly complete absence of HEAT repeats, which were shown to be involved in the binding of mTOR substrates. Real time PCR performed on 48 different human tissues demonstrated the ubiquitous presence of this splice variant. Quantification of mRNA levels in lymphoblastoid cell lines (LCL) from 56 unrelated HapMap individuals revealed that the expression of this splicing form is quite variable. One synonymous SNP, rs2289759 in exon 14, was predicted by ESEfinder to cause a significant gain/loss of SRp55 and/or SF2/ASF binding sites, and thus potentially influence splicing. This prediction was confirmed by linear regression analysis between the ratio of RAPTOR{sub v}2 to total RAPTOR mRNA levels and the SNP genotype in the above 56 individuals (r = 0.281 and P = 0.036). Moreover, the functional evaluation indicated that this splicing isoform is expected to retain the ability to bind mTOR, but is unlikely to bind mTOR substrates, hence affecting signal transduction and further cell proliferation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lapham, J.; Crothers, D.M. [Yale Univ., New Haven, CT (United States)

    1994-12-01

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

  9. A general definition and nomenclature for alternative splicing events.

    Science.gov (United States)

    Sammeth, Michael; Foissac, Sylvain; Guigó, Roderic

    2008-08-08

    Understanding the molecular mechanisms responsible for the regulation of the transcriptome present in eukaryotic cells is one of the most challenging tasks in the postgenomic era. In this regard, alternative splicing (AS) is a key phenomenon contributing to the production of different mature transcripts from the same primary RNA sequence. As a plethora of different transcript forms is available in databases, a first step to uncover the biology that drives AS is to identify the different types of reflected splicing variation. In this work, we present a general definition of the AS event along with a notation system that involves the relative positions of the splice sites. This nomenclature univocally and dynamically assigns a specific "AS code" to every possible pattern of splicing variation. On the basis of this definition and the corresponding codes, we have developed a computational tool (AStalavista) that automatically characterizes the complete landscape of AS events in a given transcript annotation of a genome, thus providing a platform to investigate the transcriptome diversity across genes, chromosomes, and species. Our analysis reveals that a substantial part--in human more than a quarter-of the observed splicing variations are ignored in common classification pipelines. We have used AStalavista to investigate and to compare the AS landscape of different reference annotation sets in human and in other metazoan species and found that proportions of AS events change substantially depending on the annotation protocol, species-specific attributes, and coding constraints acting on the transcripts. The AStalavista system therefore provides a general framework to conduct specific studies investigating the occurrence, impact, and regulation of AS.

  10. Cryptic Methane Emissions from Upland Forest Ecosystems

    Energy Technology Data Exchange (ETDEWEB)

    Megonigal, Patrick [Smithsonian Institution, Washington, DC (United States); Pitz, Scott [Johns Hopkins Univ., Baltimore, MD (United States); Smithsonian Institution, Washington, DC (United States)

    2016-04-19

    This exploratory research on Cryptic Methane Emissions from Upland Forest Ecosystems was motivated by evidence that upland ecosystems emit 36% as much methane to the atmosphere as global wetlands, yet we knew almost nothing about this source. The long-term objective was to refine Earth system models by quantifying methane emissions from upland forests, and elucidate the biogeochemical processes that govern upland methane emissions. The immediate objectives of the grant were to: (i) test the emerging paradigm that upland trees unexpectedly transpire methane, (ii) test the basic biogeochemical assumptions of an existing global model of upland methane emissions, and (iii) develop the suite of biogeochemical approaches that will be needed to advance research on upland methane emissions. We instrumented a temperate forest system in order to explore the processes that govern upland methane emissions. We demonstrated that methane is emitted from the stems of dominant tree species in temperate upland forests. Tree emissions occurred throughout the growing season, while soils adjacent to the trees consumed methane simultaneously, challenging the concept that forests are uniform sinks of methane. High frequency measurements revealed diurnal cycling in the rate of methane emissions, pointing to soils as the methane source and transpiration as the most likely pathway for methane transport. We propose the forests are smaller methane sinks than previously estimated due to stem emissions. Stem emissions may be particularly important in upland tropical forests characterized by high rainfall and transpiration, resolving differences between models and measurements. The methods we used can be effectively implemented in order to determine if the phenomenon is widespread.

  11. Alternative Spliced Transcripts as Cancer Markers

    Directory of Open Access Journals (Sweden)

    Otavia L. Caballero

    2001-01-01

    Full Text Available Eukaryotic mRNAs are transcribed as precursors containing their intronic sequences. These are subsequently excised and the exons are spliced together to form mature mRNAs. This process can lead to transcript diversification through the phenomenon of alternative splicing. Alternative splicing can take the form of one or more skipped exons, variable position of intron splicing or intron retention. The effect of alternative splicing in expanding protein repertoire might partially underlie the apparent discrepancy between gene number and the complexity of higher eukaryotes. It is likely that more than 50% form. Many cancer-associated genes, such as CD44 and WT1 are alternatively spliced. Variation of the splicing process occurs during tumor progression and may play a major role in tumorigenesis. Furthermore, alternatively spliced transcripts may be extremely useful as cancer markers, since it appears likely that there may be striking contrasts in usage of alternatively spliced transcript variants between normal and tumor tissue than in alterations in the general levels of gene expression.

  12. Geochemical evidence for cryptic sulfur cycling in salt marsh sediments

    DEFF Research Database (Denmark)

    Mills, Jennifer V.; Antler, Gilad; Turchyn, Alexandra V.

    2016-01-01

    to represent the salt marsh sediments suggests that the uptake rate of sulfate during this cryptic sulfur cycling is similar to the uptake rate of sulfate during the fastest microbial sulfate reduction that has been measured in the natural environment. The difference is that during cryptic sulfur cycling, all...... investigate sulfur cycling in salt marsh sediments from Norfolk, England where we observe high ferrous iron concentrations with no depletion of sulfate or change in the sulfur isotope ratio of that sulfate, but a 5‰ increase in the oxygen isotope ratio in sulfate, indicating that sulfate has been through...... a reductive cycle replacing its oxygen atoms. This cryptic sulfur cycle was replicated in laboratory incubations using 18O-enriched water, demonstrating that the field results do not solely result from mixing processes in the natural environment. Numerical modeling of the laboratory incubations scaled...

  13. Regulation of splicing factors by alternative splicing and NMD is conserved between kingdoms yet evolutionarily flexible.

    Science.gov (United States)

    Lareau, Liana F; Brenner, Steven E

    2015-04-01

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

  14. Posttranscriptional Regulation of Splicing Factor SRSF1 and Its Role in Cancer Cell Biology

    Directory of Open Access Journals (Sweden)

    Vânia Gonçalves

    2015-01-01

    Full Text Available Over the past decade, alternative splicing has been progressively recognized as a major mechanism regulating gene expression patterns in different tissues and disease states through the generation of multiple mRNAs from the same gene transcript. This process requires the joining of selected exons or usage of different pairs of splice sites and is regulated by gene-specific combinations of RNA-binding proteins. One archetypical splicing regulator is SRSF1, for which we review the molecular mechanisms and posttranscriptional modifications involved in its life cycle. These include alternative splicing of SRSF1 itself, regulatory protein phosphorylation events, and the role of nuclear versus cytoplasmic SRSF1 localization. In addition, we resume current knowledge on deregulated SRSF1 expression in tumors and describe SRSF1-regulated alternative transcripts with functional consequences for cancer cell biology at different stages of tumor development.

  15. Influenza A Virus Utilizes Suboptimal Splicing to Coordinate the Timing of Infection

    Directory of Open Access Journals (Sweden)

    Mark A. Chua

    2013-01-01

    Full Text Available Influenza A virus is unique as an RNA virus in that it replicates in the nucleus and undergoes splicing. With only ten major proteins, the virus must gain nuclear access, replicate, assemble progeny virions in the cytoplasm, and then egress. In an effort to elucidate the coordination of these events, we manipulated the transcript levels from the bicistronic nonstructural segment that encodes the spliced virus product responsible for genomic nuclear export. We find that utilization of an erroneous splice site ensures the slow accumulation of the viral nuclear export protein (NEP while generating excessive levels of an antagonist that inhibits the cellular response to infection. Modulation of this simple transcriptional event results in improperly timed export and loss of virus infection. Together, these data demonstrate that coordination of the influenza A virus life cycle is set by a “molecular timer” that operates on the inefficient splicing of a virus transcript.

  16. Bipartite functions of the CREB co-activators selectively direct alternative splicing or transcriptional activation.

    Science.gov (United States)

    Amelio, Antonio L; Caputi, Massimo; Conkright, Michael D

    2009-09-16

    The CREB regulated transcription co-activators (CRTCs) regulate many biological processes by integrating and converting environmental inputs into transcriptional responses. Although the mechanisms by which CRTCs sense cellular signals are characterized, little is known regarding how CRTCs contribute to the regulation of cAMP inducible genes. Here we show that these dynamic regulators, unlike other co-activators, independently direct either pre-mRNA splice-site selection or transcriptional activation depending on the cell type or promoter context. Moreover, in other scenarios, the CRTC co-activators coordinately regulate transcription and splicing. Mutational analyses showed that CRTCs possess distinct functional domains responsible for regulating either pre-mRNA splicing or transcriptional activation. Interestingly, the CRTC1-MAML2 oncoprotein lacks the splicing domain and is incapable of altering splice-site selection despite robustly activating transcription. The differential usage of these distinct domains allows CRTCs to selectively mediate multiple facets of gene regulation, indicating that co-activators are not solely restricted to coordinating alternative splicing with increase in transcriptional activity.

  17. Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development.

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    Nathalie Schmieg

    Full Text Available Kidins220/ARMS is a transmembrane protein playing a crucial role in neuronal and cardiovascular development. Kidins220/ARMS is a downstream target of neurotrophin receptors and interacts with several signalling and trafficking factors. Through computational modelling, we found two potential sites for alternative splicing of Kidins220/ARMS. The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33. Here we describe the conserved occurrence of several Kidins220/ARMS splice isoforms at RNA and protein levels. Kidins220/ARMS splice isoforms display spatio-temporal regulation during development with distinct patterns in different neuronal populations. Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript. Remarkably, alternative terminal exon splicing generates Kidins220/ARMS variants with distinct cellular localisation: Kidins220/ARMS containing exon 32 is targeted to the plasma membrane and neurite tips, whereas Kidins220/ARMS without exon 33 mainly clusters the full-length protein in a perinuclear intracellular compartment in PC12 cells and primary neurons, leading to a change in neurotrophin receptor expression. Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development.

  18. Computational Analysis of an Evolutionarily Conserved VertebrateMuscle Alternative Splicing Program

    Energy Technology Data Exchange (ETDEWEB)

    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

    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.

  19. A novel splicing mutation alters DSPP transcription and leads to dentinogenesis imperfecta type II.

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    Jun Zhang

    Full Text Available Dentinogenesis imperfecta (DGI type II is an autosomal dominant disease characterized by a serious disorders in teeth. Mutations of dentin sialophosphoprotein (DSPP gene were revealed to be the causation of DGI type II (DGI-II. In this study, we identified a novel mutation (NG_011595.1:g.8662T>C, c.135+2T>C lying in the splice donor site of intron 3 of DSPP gene in a Chinese Han DGI-II pedigree. It was found in all affected subjects but not in unaffected ones or other unrelated healthy controls. The function of the mutant DSPP gene, which was predicted online and subsequently confirmed by in vitro splicing analysis, was the loss of splicing of intron 3, leading to the extended length of DSPP mRNA. For the first time, the functional non-splicing of intron was revealed in a novel DSPP mutation and was considered as the causation of DGI-II. It was also indicated that splicing was of key importance to the function of DSPP and this splice donor site might be a sensitive mutation hot spot. Our findings combined with other reports would facilitate the genetic diagnosis of DGI-II, shed light on its gene therapy and help to finally conquer human diseases.

  20. A novel splicing mutation alters DSPP transcription and leads to dentinogenesis imperfecta type II.

    Science.gov (United States)

    Zhang, Jun; Wang, Jiucun; Ma, Yanyun; Du, Wenqi; Zhao, Siyang; Zhang, Zuowei; Zhang, Xiaojiao; Liu, Yue; Xiao, Huasheng; Wang, Hongyan; Jin, Li; Liu, Jie

    2011-01-01

    Dentinogenesis imperfecta (DGI) type II is an autosomal dominant disease characterized by a serious disorders in teeth. Mutations of dentin sialophosphoprotein (DSPP) gene were revealed to be the causation of DGI type II (DGI-II). In this study, we identified a novel mutation (NG_011595.1:g.8662T>C, c.135+2T>C) lying in the splice donor site of intron 3 of DSPP gene in a Chinese Han DGI-II pedigree. It was found in all affected subjects but not in unaffected ones or other unrelated healthy controls. The function of the mutant DSPP gene, which was predicted online and subsequently confirmed by in vitro splicing analysis, was the loss of splicing of intron 3, leading to the extended length of DSPP mRNA. For the first time, the functional non-splicing of intron was revealed in a novel DSPP mutation and was considered as the causation of DGI-II. It was also indicated that splicing was of key importance to the function of DSPP and this splice donor site might be a sensitive mutation hot spot. Our findings combined with other reports would facilitate the genetic diagnosis of DGI-II, shed light on its gene therapy and help to finally conquer human diseases.

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

    Science.gov (United States)

    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

    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

  2. No Evidence for Temporal Variation in a Cryptic Species Community of Freshwater Amphipods of the Hyalella azteca Species Complex

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    Christian Nozais

    2011-08-01

    Full Text Available The co-occurrence of cryptic species of Hyalella amphipods is a challenge to our traditional views of how species assemble. Since these species have similar morphologies, it is not evident that they have developed phenotypic differences that would allow them to occupy different ecological niches. We examined the structure of a community of Hyalella amphipods in the littoral zone of a boreal lake to verify if temporal variation was present in relative abundances. Morphological and molecular analyses using the mitochondrial cytochrome c oxidase I (COI gene enabled us to detect three cryptic species at the study site. No temporal variation was observed in the community, as one cryptic species was always more abundant than the two others. The relative abundances of each species in the community appeared constant at least for the open-water season, both for adult and juvenile amphipods. Niche differences are still to be found among these species, but it is suggested that migration from nearby sites may be an important factor explaining the species co-occurrence.

  3. Antifungal susceptibility profile of cryptic species of Aspergillus.

    Science.gov (United States)

    Alastruey-Izquierdo, Ana; Alcazar-Fuoli, Laura; Cuenca-Estrella, Manuel

    2014-12-01

    The use of molecular tools has led to the description of new cryptic species among different Aspergillus species complexes. Their frequency in the clinical setting has been reported to be between 10 and 15%. The susceptibility to azoles and amphotericin B of many of these species is low, and some of them, such as Aspergillus calidoustus or Aspergillus lentulus, are considered multi-resistant. The changing epidemiology, the frequency of cryptic species, and the different susceptibility profiles make antifungal susceptibility testing an important tool to identify the optimal antifungal agent to treat the infections caused by these species.

  4. Hidden Diversity in Sardines: Genetic and Morphological Evidence for Cryptic Species in the Goldstripe Sardinella, Sardinella gibbosa (Bleeker, 1849)

    Science.gov (United States)

    Thomas, Rey C.; Willette, Demian A.; Carpenter, Kent E.; Santos, Mudjekeewis D.

    2014-01-01

    Cryptic species continue to be uncovered in many fish taxa, posing challenges for fisheries conservation and management. In Sardinella gibbosa, previous investigations revealed subtle intra-species variations, resulting in numerous synonyms and a controversial taxonomy for this sardine. Here, we tested for cryptic diversity within S. gibbosa using genetic data from two mitochondrial and one nuclear gene regions of 248 individuals of S. gibbosa, collected from eight locations across the Philippine archipelago. Deep genetic divergence and subsequent clustering was consistent across both mitochondrial and nuclear markers. Clade distribution is geographically limited: Clade 1 is widely distributed in the central Philippines, while Clade 2 is limited to the northernmost sampling site. In addition, morphometric analyses revealed a unique head shape that characterized each genetic clade. Hence, both genetic and morphological evidence strongly suggests a hidden diversity within this common and commercially-important sardine. PMID:24416271

  5. Hidden diversity in sardines: genetic and morphological evidence for cryptic species in the goldstripe sardinella, Sardinella gibbosa (Bleeker, 1849.

    Directory of Open Access Journals (Sweden)

    Rey C Thomas

    Full Text Available Cryptic species continue to be uncovered in many fish taxa, posing challenges for fisheries conservation and management. In Sardinella gibbosa, previous investigations revealed subtle intra-species variations, resulting in numerous synonyms and a controversial taxonomy for this sardine. Here, we tested for cryptic diversity within S. gibbosa using genetic data from two mitochondrial and one nuclear gene regions of 248 individuals of S. gibbosa, collected from eight locations across the Philippine archipelago. Deep genetic divergence and subsequent clustering was consistent across both mitochondrial and nuclear markers. Clade distribution is geographically limited: Clade 1 is widely distributed in the central Philippines, while Clade 2 is limited to the northernmost sampling site. In addition, morphometric analyses revealed a unique head shape that characterized each genetic clade. Hence, both genetic and morphological evidence strongly suggests a hidden diversity within this common and commercially-important sardine.

  6. Protein trans-splicing of an atypical split intein showing structural flexibility and cross-reactivity.

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    Huiling Song

    Full Text Available Inteins catalyze a protein splicing reaction to excise the intein from a precursor protein and join the flanking sequences (exteins with a peptide bond. In a split intein, the intein fragments (I(N and I(C can reassemble non-covalently to catalyze a trans-splicing reaction that joins the exteins from separate polypeptides. An atypical split intein having a very small I(N and a large I(C is particularly useful for joining synthetic peptides with recombinant proteins, which can be a generally useful method of introducing site-specific chemical labeling or modifications into proteins. However, a large I(C derived from an Ssp DnaX intein was found recently to undergo spontaneous C-cleavage, which raised questions regarding its structure-function and ability to trans-splice. Here, we show that this I(C could undergo trans-splicing in the presence of I(N, and the trans-splicing activity completely suppressed the C-cleavage activity. We also found that this I(C could trans-splice with small I(N sequences derived from two other inteins, showing a cross-reactivity of this atypical split intein. Furthermore, we found that this I(C could trans-splice even when the I(N sequence was embedded in a nearly complete intein sequence, suggesting that the small I(N could project out of the central pocket of the intein to become accessible to the I(C. Overall, these findings uncovered a new atypical split intein that can be valuable for peptide-protein trans-splicing, and they also revealed an interesting structural flexibility and cross-reactivity at the active site of this intein.

  7. Please mind the gap - Visual census and cryptic biodiversity assessment at central Red Sea coral reefs.

    Science.gov (United States)

    Pearman, John K; Anlauf, Holger; Irigoien, Xabier; Carvalho, Susana

    2016-07-01

    Coral reefs harbor the most diverse assemblages in the ocean, however, a large proportion of the diversity is cryptic and, therefore, undetected by standard visual census techniques. Cryptic and exposed communities differ considerably in species composition and ecological function. This study compares three different coral reef assessment protocols: i) visual benthic reef surveys: ii) visual census of Autonomous Reef Monitoring Structures (ARMS) plates; and iii) metabarcoding techniques of the ARMS (including sessile, 106-500 μm and 500-2000 μm size fractions), that target the cryptic and exposed communities of three reefs in the central Red Sea. Visual census showed a dominance of Cnidaria (Anthozoa) and Rhodophyta on the reef substrate, while Porifera, Bryozoa and Rhodophyta were the most abundant groups on the ARMS plates. Metabarcoding, targeting the 18S rRNA gene, significantly increased estimates of the species diversity (p reefs. Furthermore, metabarcoding detected microbial eukaryotic groups such as Syndiniophyceae, Mamiellophyceae and Bacillariophyceae as relevant components of the sessile fraction. ANOSIM analysis showed that the three reef sites showed no differences based on the visual census data. Metabarcoding showed a higher sensitivity for identifying differences between reef communities at smaller geographic scales than standard visual census techniques as significant differences in the assemblages were observed amongst the reefs. Comparison of the techniques showed no similar patterns for the visual techniques while the metabarcoding of the ARMS showed similar patterns amongst fractions. Establishing ARMS as a standard tool in reef monitoring will not only advance our understanding of local processes and ecological community response to environmental changes, as different faunal components will provide complementary information but also improve the estimates of biodiversity in coral reef benthic communities. This study lays the foundations

  8. Single nucleotide polymorphism-based validation of exonic splicing enhancers.

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    William G Fairbrother

    2004-09-01

    Full Text Available Because deleterious alleles arising from mutation are filtered by natural selection, mutations that create such alleles will be underrepresented in the set of common genetic variation existing in a population at any given time. Here, we describe an approach based on this idea called VERIFY (variant elimination reinforces functionality, which can be used to assess the extent of natural selection acting on an oligonucleotide motif or set of motifs predicted to have biological activity. As an application of this approach, we analyzed a set of 238 hexanucleotides previously predicted to have exonic splicing enhancer (ESE activity in human exons using the relative enhancer and silencer classification by unanimous enrichment (RESCUE-ESE method. Aligning the single nucleotide polymorphisms (SNPs from the public human SNP database to the chimpanzee genome allowed inference of the direction of the mutations that created present-day SNPs. Analyzing the set of SNPs that overlap RESCUE-ESE hexamers, we conclude that nearly one-fifth of the mutations that disrupt predicted ESEs have been eliminated by natural selection (odds ratio = 0.82 +/- 0.05. This selection is strongest for the predicted ESEs that are located near splice sites. Our results demonstrate a novel approach for quantifying the extent of natural selection acting on candidate functional motifs and also suggest certain features of mutations/SNPs, such as proximity to the splice site and disruption or alteration of predicted ESEs, that should be useful in identifying variants that might cause a biological phenotype.

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

    Science.gov (United States)

    Pollastro, Pasquale; Rampone, Salvatore

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

  10. Temperature-sensitive splicing is an important molecular regulation mechanism of thermosensitive genie male sterility in rice

    Institute of Scientific and Technical Information of China (English)

    CHEN RongZhi; PAN YuFang; WANG Yang; ZHU LiLi; HE GuangCun

    2009-01-01

    Photoperiod and temperature-sensitive genetic male sterility (PGMS and TGMS) plants have a number of desirable characteristics for hybrid production. Two-line hybrids developed using the PGMS/TGMS system now account for a large proportion of rice production in China. In this paper, we summarize recent advances on molecular regulation mechanisms and genetics of PGMS/TGMS in rice. We suggest that temperature-sensitive splicing, an important posttranscriptional regulatory mechanism in modulating gene expression and eventually development and differentiation, is also an important molecular regulation mechanism of TGMS in rice. We review those factors involved in temperature-sensitive splicing like cis splice site, snRNA, trans pre-mRNA splicing protein and SR proteins, and delineate that splicing could be regulated by a complex cell signaling pathway. These might shed light on other unknown molecular PGMS/TGMS mechanisms.

  11. South Polar Cryptic Terrain in Early Spring

    Science.gov (United States)

    2007-01-01

    This image of the south polar region of Mars was taken by the Compact Reconnaissance Imaging Spectrometer for Mars (CRISM) at 1557 UTC (10:57 a.m. EST) on Feb. 10, 2007, near 77.55 degrees south latitude, 131.98 degrees east longitude. CRISM's image was taken in 544 colors covering 0.36-3.92 micrometers, and shows features as small as 30 meters (98 feet) across. The region covered is just over 9 kilometers (5.6 miles) wide at its narrowest point, and is one of several in which CRISM is monitoring the evaporation (or 'sublimation') of the seasonal frost cap. The Martian south polar seasonal cap consists of carbon dioxide ice and frost, whose sublimation in the Martian spring creates a variety of features unlike anything in Earth's circumpolar regions. Part of the cap known as the 'cryptic region' is so cold that it must be covered with carbon dioxide frost, but it is also unexpectedly low in brightness and exhibits a variety of unusual dark blotches. Many scientists believe that carbon dioxide gas trapped below the sublimating ice is released in bursts, which carry along dust that gradually darkens the ice. One idea is that geyser-like dust eruptions form the dark blotches, and that the blotches grow, coalesce, and eventually hide the frost under a thin layer of dust. This image was taken shortly after sunrise with the Sun only about five degrees above the horizon. The left version shows brightness of the surface at 1.3 micrometers. The right version shows strength of an absorption band due to carbon dioxide frost at 1.435 micrometers; brighter areas have a stronger absorption and more carbon dioxide frost. However, even the darkest areas still have frost. The correlation between brightness and carbon dioxide frost abundance is striking, supporting the idea that the frost is being darkened by dust. Frost in the upper right corner shows the expected dark blotches, whereas the frost over the rest of the image is more uniformly dark, hinting that another darkening

  12. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.

    Science.gov (United States)

    Sveen, A; Kilpinen, S; Ruusulehto, A; Lothe, R A; Skotheim, R I

    2016-05-12

    Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations.

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

    KAUST Repository

    Cui, Peng

    2014-01-07

    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. 2014 Cui et al.; licensee BioMed Central Ltd.

  14. Using Profiles Based on Nucleotide Hydrophobicity to Define Essential Regions for Splicing

    Directory of Open Access Journals (Sweden)

    Galina Boldina, Anatoly Ivashchenko, Mireille Régnier

    2009-01-01

    Full Text Available The splice-site sequences of U2-type introns are highly degenerate, so many different sequences can function as U2-type splice sites. Using our new profiles based on hydrophobicity properties we pointed out specific properties for regions surrounding splice sites. We built a set T of flanking regions of genes with 1-3 introns from 21st and 22nd chromosomes extracted from GenBank to define positions having conserved properties, namely hydrophobicity, that are potentially essential for recognition by spliceosome. GT–AG introns exist in U2 and U12-types. Therefore, intron type cannot be simply determined by the dinucleotide termini. We attempted to distinguish U2 and U12-types introns with help of hydrophobicity profiles on sets of spice sites for U2 or U12-type introns extracted from SpliceRack database. The positions given by our method, which may be important for recognition by spliceosome, were compared to the nucleotide consensus provided by a classical method, Pictogram. We showed that there is a similarity of hydrophobicity profiles inside intron types. On one hand, GT–AG and GC–AG introns belonging to U2-type have resembling hydrophobicity profiles as well as AT–AC and GT–AG introns belonging to U12-type. On the other hand, hydrophobicity profiles of U2 and U12-types GT–AG introns are completely different. We suggest that hydrophobicity profiles facilitate definition of intron type, distinguishing U2 and U12 intron types and can be used to build programs to search splice site and to evaluate their strength. Therefore, our study proves that hydrophobicity profiles are informative method providing insights into mechanisms of splice sites recognition.

  15. Alcoholism and Alternative Splicing of Candidate Genes

    Directory of Open Access Journals (Sweden)

    Toshikazu Sasabe

    2010-03-01

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

  16. Tumor microenvironment–associated modifications of alternative splicing

    Science.gov (United States)

    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

    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

  17. A widespread and unusual RNA trans-splicing type in dinoflagellate mitochondria.

    Science.gov (United States)

    Jackson, Christopher J; Waller, Ross F

    2013-01-01

    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.

  18. A widespread and unusual RNA trans-splicing type in dinoflagellate mitochondria.

    Directory of Open Access Journals (Sweden)

    Christopher J Jackson

    Full Text Available 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.

  19. Live-Cell Visualization of Pre-mRNA Splicing with Single-Molecule Sensitivity

    Directory of Open Access Journals (Sweden)

    Robert M. Martin

    2013-09-01

    Full Text Available Removal of introns from pre-messenger RNAs (pre-mRNAs via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20–30 s. Furthermore, we show that replacing the weak polypyrimidine (Py tract in mouse immunoglobulin μ (IgM pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min−1 and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context.

  20. Differential splicing of exon 5 of the Wilms tumour (WTI) gene.

    Science.gov (United States)

    Renshaw, J; King-Underwood, L; Pritchard-Jones, K

    1997-08-01

    The WTI gene encodes a developmentally regulated transcription factor whose function is altered by alternative splicing at two sites: the 17 amino acids of exon 5, whose functional effects are ill-defined, and the 3 amino acids (KTS) between exons 9 and 10, which determine sequence-specific DNA binding and nuclear localisation. Germline mutations, which prevent normal KTS splicing, can underlie the Denys-Drash syndrome, and disruptions of splicing of exon 5 may occur in Wilms tumours. We analysed by reverse transcriptase polymerase chain reaction (RT-PCR) amplification the relative ratios of the four splice variants of WTI mRNA in normal and tumour tissues and found tissue-specific, developmental stage-specific, and species-specific differences in the splicing of exon 5 but not of KTS. We found no evidence for disrupted splicing in acute leukaemias or gonadal tumours. The significance of these findings is discussed, and the possibility is raised that WTI may orchestrate the appropriate response to growth and differentiation factor signalling, mediated by alterations in the relative levels of exon 5 containing WTI isoforms.

  1. New discoveries of old SON: a link between RNA splicing and cancer.

    Science.gov (United States)

    Hickey, Christopher J; Kim, Jung-Hyun; Ahn, Eun-Young Erin

    2014-02-01

    The SON protein is a ubiquitously expressed DNA- and RNA-binding protein primarily localized to nuclear speckles. Although several early studies implicated SON in DNA-binding, tumorigenesis and apoptosis, functional significance of this protein had not been recognized until recent studies discovered SON as a novel RNA splicing co-factor. During constitutive RNA splicing, SON ensures efficient intron removal from the transcripts containing suboptimal splice sites. Importantly, SON-mediated splicing is required for proper processing of selective transcripts related to cell cycle, microtubules, centrosome maintenance, and genome stability. Moreover, SON regulates alternative splicing of RNAs from the genes involved in apoptosis and epigenetic modification. In addition to the role in RNA splicing, SON has an ability to suppress transcriptional activation at certain promoter/enhancer DNA sequences. Considering the multiple SON target genes which are directly involved in cell proliferation, genome stability and chromatin modifications, SON is an emerging player in gene regulation during cancer development and progression. Here, we summarize available information from several early studies on SON, and highlight recent discoveries describing molecular mechanisms of SON-mediated gene regulation. We propose that our future effort on better understanding of diverse SON functions would reveal novel targets for cancer therapy.

  2. Spliced

    DEFF Research Database (Denmark)

    Addison, Courtney Page

    2017-01-01

    Human gene therapy (HGT) aims to cure disease by inserting or editing the DNA of patients with genetic conditions. Since foundational genetic techniques came into use in the 1970s, the field has developed to the point that now three therapies have market approval, and over 1800 clinical trials have...

  3. A bridge too far: dispersal barriers and cryptic speciation in an Arabian Peninsula grouper (Cephalopholis hemistiktos)

    KAUST Repository

    Priest, Mark

    2015-12-12

    Aim: We use genetic and age-based analyses to assess the evidence for a biogeographical barrier to larval dispersal in the yellowfin hind, Cephalopholis hemistiktos, a commercially important species found across the Arabian Peninsula. Location: Red Sea, Gulf of Aden, Gulf of Oman and Arabian Gulf. Methods: Mitochondrial DNA cytochrome-c oxidase subunit-I and nuclear DNA (S7) sequences were obtained for C. hemistiktos sampled throughout its distributional range. Phylogeographical and population-level analyses were used to assess patterns of genetic structure and to identify barriers to dispersal. Concurrently, age-based demographic analyses using otoliths determined differences in growth and longevity between regions. Results: Our analyses revealed significant genetic structure congruent with growth parameter differences observed across sampling sites, suggesting cryptic speciation between populations in the Red Sea and Gulf of Aden versus the Gulf of Oman and Arabian Gulf. Coalescence analyses indicated these two regions have been isolated for > 800,000 years. Main conclusions: Our results indicate historical disruption to gene flow and a contemporary dispersal barrier in the Arabian Sea, which C. hemistiktos larvae are unable to effectively traverse. This provides yet another example of a (cryptic) species with high dispersive potential whose range is delimited by a lack of suitable habitat between locations or an inability to successfully recruit at the range edge. © 2015 John Wiley & Sons Ltd.

  4. A human PrM antibody that recognizes a novel cryptic epitope on dengue E glycoprotein.

    Science.gov (United States)

    Chan, Annie Hoi Yi; Tan, Hwee Cheng; Chow, Angelia Yee; Lim, Angeline Pei Chiew; Lok, Shee Mei; Moreland, Nicole J; Vasudevan, Subhash G; MacAry, Paul A; Ooi, Eng Eong; Hanson, Brendon J

    2012-01-01

    Dengue virus (DENV) is a major mosquito-borne pathogen infecting up to 100 million people each year; so far no effective treatment or vaccines are available. Recently, highly cross-reactive and infection-enhancing pre-membrane (prM)-specific antibodies were found to dominate the anti-DENV immune response in humans, raising concern over vaccine candidates that contain native dengue prM sequences. In this study, we have isolated a broadly cross-reactive prM-specific antibody, D29, during a screen with a non-immunized human Fab-phage library against the four serotypes of DENV. The antibody is capable of restoring the infectivity of virtually non-infectious immature DENV (imDENV) in FcγR-bearing K562 cells. Remarkably, D29 also cross-reacted with a cryptic epitope on the envelope (E) protein located to the DI/DII junction as evidenced by site-directed mutagenesis. This cryptic epitope, while inaccessible to antibody binding in a native virus particle, may become exposed if E is not properly folded. These findings suggest that generation of anti-prM antibodies that enhance DENV infection may not be completely avoided even with immunization strategies employing E protein alone or subunits of E proteins.

  5. Cryptic Transcription and Early Termination in the Control of Gene Expression

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    Jessie Colin

    2011-01-01

    Full Text Available Recent studies on yeast transcriptome have revealed the presence of a large set of RNA polymerase II transcripts mapping to intergenic and antisense regions or overlapping canonical genes. Most of these ncRNAs (ncRNAs are subject to termination by the Nrd1-dependent pathway and rapid degradation by the nuclear exosome and have been dubbed cryptic unstable transcripts (CUTs. CUTs are often considered as by-products of transcriptional noise, but in an increasing number of cases they play a central role in the control of gene expression. Regulatory mechanisms involving expression of a CUT are diverse and include attenuation, transcriptional interference, and alternative transcription start site choice. This review focuses on the impact of cryptic transcription on gene expression, describes the role of the Nrd1-complex as the main actor in preventing nonfunctional and potentially harmful transcription, and details a few systems where expression of a CUT has an essential regulatory function. We also summarize the most recent studies concerning other types of ncRNAs and their possible role in regulation.

  6. Contrasting biological features in morphologically cryptic Mediterranean sponges

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    Leire Garate

    2017-06-01

    Full Text Available Sponges are key organisms in the marine benthos where they play essential roles in ecological processes such as creating new niches, competition for resources, and organic matter recycling. Despite the increasing number of taxonomical studies, many sponge species remain hidden, whether unnoticed or cryptic. The occurrence of cryptic species may confound ecological studies by underestimating biodiversity. In this study, we monitored photographically growth, fusions, fissions, and survival of two morphologically cryptic species Hemimycale mediterranea Uriz, Garate & Agell, 2017 and H. columella (Bowerbank, 1874. Additionally, we characterized the main environmental factors of the corresponding species habitats, trying to ascertain whether some abiotic factors were correlated with the distribution of these species. Sponge monitoring was performed monthly. Seawater samples were collected the same monitoring days in the vicinity of the target sponges. Results showed contrasting growth and survival patterns for each species: H. mediterranea totally disappeared after larval release while 64% of individuals of H. columella survived the entire two years we monitored. The species also differed in the number of fissions and fusions. These events were evenly distributed throughout the year in the H. mediterranea population but concentrated in cold months in H. columella. No measured environmental factor correlated with H. mediterranea growth rates, while temperature and dissolved organic nitrogen were negatively correlated with H. columella growth rates. The strong differences in depth distribution, survival, growth, fusions, and fissions found between these two cryptic species, highlights the importance of untangling cryptic species before ecological studies are performed in particular when these species share geographical distribution.

  7. Splicosomal and serine and arginine-rich splicing factors as targets for TGF-β

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    Hallgren Oskar

    2012-04-01

    Full Text Available Abstract Background Transforming growth factor-β1 (TGF-β1 is a potent regulator of cell growth and differentiation. TGF-β1 has been shown to be a key player in tissue remodeling processes in a number of disease states by inducing expression of extracellular matrix proteins. In this study a quantitative proteomic analysis was undertaken to investigate if TGF-β1 contributes to tissue remodeling by mediating mRNA splicing and production of alternative isoforms of proteins. Methodology/Principal findings The expression of proteins involved in mRNA splicing from TGF-β1-stimulated lung fibroblasts was compared to non-stimulated cells by employing isotope coded affinity tag (ICATTM reagent labeling and tandem mass spectrometry. A total of 1733 proteins were identified and quantified with a relative standard deviation of 11% +/− 8 from enriched nuclear fractions. Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection. In addition, TGF-β1 was observed to alter the relative expression of splicing proteins that may be important for alternative splicing of fibronectin. Specifically, TGF-β1 significantly induced expression of SRp20, and reduced the expression of SRp30C, which has been suggested to be a prerequisite for generation of alternatively spliced fibronectin. The induction of SRp20 was further confirmed by western blot and immunofluorescence. Conclusions The results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts. This may have an impact on the production of alternative isoforms of matrix proteins and can therefore be an important factor in tissue remodeling and disease progression.

  8. A methyl transferase links the circadian clock to the regulation of alternative splicing.

    Science.gov (United States)

    Sanchez, Sabrina E; Petrillo, Ezequiel; Beckwith, Esteban J; Zhang, Xu; Rugnone, Matias L; Hernando, C Esteban; Cuevas, Juan C; Godoy Herz, Micaela A; Depetris-Chauvin, Ana; Simpson, Craig G; Brown, John W S; Cerdán, Pablo D; Borevitz, Justin O; Mas, Paloma; Ceriani, M Fernanda; Kornblihtt, Alberto R; Yanovsky, Marcelo J

    2010-11-04

    Circadian rhythms allow organisms to time biological processes to the most appropriate phases of the day-night cycle. Post-transcriptional regulation is emerging as an important component of circadian networks, but the molecular mechanisms linking the circadian clock to the control of RNA processing are largely unknown. Here we show that PROTEIN ARGININE METHYL TRANSFERASE 5 (PRMT5), which transfers methyl groups to arginine residues present in histones and Sm spliceosomal proteins, links the circadian clock to the control of alternative splicing in plants. Mutations in PRMT5 impair several circadian rhythms in Arabidopsis thaliana and this phenotype is caused, at least in part, by a strong alteration in alternative splicing of the core-clock gene PSEUDO RESPONSE REGULATOR 9 (PRR9). Furthermore, genome-wide studies show that PRMT5 contributes to the regulation of many pre-messenger-RNA splicing events, probably by modulating 5'-splice-site recognition. PRMT5 expression shows daily and circadian oscillations, and this contributes to the mediation of the circadian regulation of expression and alternative splicing of a subset of genes. Circadian rhythms in locomotor activity are also disrupted in dart5-1, a mutant affected in the Drosophila melanogaster PRMT5 homologue, and this is associated with alterations in splicing of the core-clock gene period and several clock-associated genes. Our results demonstrate a key role for PRMT5 in the regulation of alternative splicing and indicate that the interplay between the circadian clock and the regulation of alternative splicing by PRMT5 constitutes a common mechanism that helps organisms to synchronize physiological processes with daily changes in environmental conditions.

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

    Science.gov (United States)

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

    2014-04-01

    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)1α, Nrxn1β, Nrxn2β, Nrxn3α, and Nrxn3β 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 Nrxn1α and Nrxn3α (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-α, 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 α-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.

  10. Reproductive isolation and ecological niche partition among larvae of the morphologically cryptic sister species Chironomus riparius and C. piger.

    Directory of Open Access Journals (Sweden)

    Markus Pfenninger

    Full Text Available BACKGROUND: One of the central issues in ecology is the question what allows sympatric occurrence of closely related species in the same general area? The non-biting midges Chironomus riparius and C. piger, interbreeding in the laboratory, have been shown to coexist frequently despite of their close relatedness, similar ecology and high morphological similarity. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate factors shaping niche partitioning of these cryptic sister species, we explored the actual degree of reproductive isolation in the field. Congruent results from nuclear microsatellite and mitochondrial haplotype analyses indicated complete absence of interspecific gene-flow. Autocorrelation analysis showed a non-random spatial distribution of the two species. Though not dispersal limited at the scale of the study area, the sister species occurred less often than expected at the same site, indicating past or present competition. Correlation and multiple regression analyses suggested the repartition of the available habitat along water chemistry gradients (nitrite, conductivity, CaCO(3, ultimately governed by differences in summer precipitation regime. CONCLUSIONS: We show that these morphologically cryptic sister species partition their niches due to a certain degree of ecological distinctness and total reproductive isolation in the field. The coexistence of these species provides a suitable model system for the investigation of factors shaping the distribution of closely related, cryptic species.

  11. Why Do Cryptic Species Tend Not to Co-Occur? A Case Study on Two Cryptic Pairs of Butterflies

    Science.gov (United States)

    Vodă, Raluca; Dapporto, Leonardo; Dincă, Vlad; Vila, Roger

    2015-01-01

    As cryptic diversity is being discovered, mostly thanks to advances in molecular techniques, it is becoming evident that many of these taxa display parapatric distributions in mainland and that they rarely coexist on islands. Genetic landscapes, haplotype networks and ecological niche modeling analyses were performed for two pairs of non-sister cryptic butterfly species, Aricia agestis-A. cramera and Polyommatus icarus—P. celina (Lycaenidae), to specifically assess non-coexistence on western Mediterranean islands, and to test potential causes producing such chequered distribution patterns. We show that the morphologically and ecologically equivalent pairs of species do not coexist on any of the studied islands, although nearly all islands are colonized by one of them. According to our models, the cryptic pairs displayed marked climatic preferences and ‘precipitation during the driest quarter’ was recovered as the most important climatic determinant. However, neither dispersal capacity, nor climatic or ecological factors fully explain the observed distributions across particular sea straits, and the existence of species interactions resulting in mutual exclusion is suggested as a necessary hypothesis. Given that the studied species are habitat generalists, feeding on virtually unlimited resources, we propose that reproductive interference, together with climatic preferences, sustain density-dependent mechanisms like “founder takes all” and impede coexistence on islands. Chequered distributions among cryptic taxa, both sister and non-sister, are common in butterflies, suggesting that the phenomenon revealed here could be important in determining biodiversity patterns. PMID:25692577

  12. Why do cryptic species tend not to co-occur? A case study on two cryptic pairs of butterflies.

    Directory of Open Access Journals (Sweden)

    Raluca Vodă

    Full Text Available As cryptic diversity is being discovered, mostly thanks to advances in molecular techniques, it is becoming evident that many of these taxa display parapatric distributions in mainland and that they rarely coexist on islands. Genetic landscapes, haplotype networks and ecological niche modeling analyses were performed for two pairs of non-sister cryptic butterfly species, Aricia agestis-A. cramera and Polyommatus icarus-P. celina (Lycaenidae, to specifically assess non-coexistence on western Mediterranean islands, and to test potential causes producing such chequered distribution patterns. We show that the morphologically and ecologically equivalent pairs of species do not coexist on any of the studied islands, although nearly all islands are colonized by one of them. According to our models, the cryptic pairs displayed marked climatic preferences and 'precipitation during the driest quarter' was recovered as the most important climatic determinant. However, neither dispersal capacity, nor climatic or ecological factors fully explain the observed distributions across particular sea straits, and the existence of species interactions resulting in mutual exclusion is suggested as a necessary hypothesis. Given that the studied species are habitat generalists, feeding on virtually unlimited resources, we propose that reproductive interference, together with climatic preferences, sustain density-dependent mechanisms like "founder takes all" and impede coexistence on islands. Chequered distributions among cryptic taxa, both sister and non-sister, are common in butterflies, suggesting that the phenomenon revealed here could be important in determining biodiversity patterns.

  13. Why do cryptic species tend not to co-occur? A case study on two cryptic pairs of butterflies.

    Science.gov (United States)

    Vodă, Raluca; Dapporto, Leonardo; Dincă, Vlad; Vila, Roger

    2015-01-01

    As cryptic diversity is being discovered, mostly thanks to advances in molecular techniques, it is becoming evident that many of these taxa display parapatric distributions in mainland and that they rarely coexist on islands. Genetic landscapes, haplotype networks and ecological niche modeling analyses were performed for two pairs of non-sister cryptic butterfly species, Aricia agestis-A. cramera and Polyommatus icarus-P. celina (Lycaenidae), to specifically assess non-coexistence on western Mediterranean islands, and to test potential causes producing such chequered distribution patterns. We show that the morphologically and ecologically equivalent pairs of species do not coexist on any of the studied islands, although nearly all islands are colonized by one of them. According to our models, the cryptic pairs displayed marked climatic preferences and 'precipitation during the driest quarter' was recovered as the most important climatic determinant. However, neither dispersal capacity, nor climatic or ecological factors fully explain the observed distributions across particular sea straits, and the existence of species interactions resulting in mutual exclusion is suggested as a necessary hypothesis. Given that the studied species are habitat generalists, feeding on virtually unlimited resources, we propose that reproductive interference, together with climatic preferences, sustain density-dependent mechanisms like "founder takes all" and impede coexistence on islands. Chequered distributions among cryptic taxa, both sister and non-sister, are common in butterflies, suggesting that the phenomenon revealed here could be important in determining biodiversity patterns.

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

    Science.gov (United States)

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

    2017-01-01

    Although the efforts to understand the genetic basis of cancer allowed advances in diagnosis and therapy, little is known about other molecular bases. Splicing is a key event in gene expression, controlling the excision of introns decoded inside genes and being responsible for 80% of the proteome amplification through events of alternative splicing. Growing data from the last decade point to deregulation of splicing events as crucial in carcinogenesis and tumor progression. Several alterations in splicing events were observed in cancer, caused by either missexpression of or detrimental mutations in some splicing factors, and appear to be critical in carcinogenesis and key events during tumor progression. Notwithstanding, it is difficult to determine whether it is a cause or consequence of cancer and/or tumorigenesis. Most reviews focus on the generated isoforms of deregulated splicing pattern, while others mainly summarize deregulated splicing factors observed in cancer. In this review, events associated with carcinogenesis and tumor progression mainly, and epithelial-to-mesenchymal transition, which is also implicated in alternative splicing regulation, will be progressively discussed in the light of a new perspective, suggesting that splicing deregulation mediates cell reprogramming in tumor progression by an imperfect shift of the splice program. © 2016 S. Karger AG, Basel.

  15. Behind the Scene Role of Conserved Threonine in Intein Splicing

    Science.gov (United States)

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

    2012-02-01

    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.

  16. HRP-2, the Caenorhabditis elegans homolog of mammalian heterogeneous nuclear ribonucleoproteins Q and R, is an alternative splicing factor that binds to UCUAUC splicing regulatory elements.

    Science.gov (United States)

    Kabat, Jennifer L; Barberan-Soler, Sergio; Zahler, Alan M

    2009-10-16

    Alternative splicing is regulated by cis sequences in the pre-mRNA that serve as binding sites for trans-acting alternative splicing factors. In a previous study, we used bioinformatics and molecular biology to identify and confirm that the intronic hexamer sequence UCUAUC is a nematode alternative splicing regulatory element. In this study, we used RNA affinity chromatography to identify trans factors that bind to this sequence. HRP-2, the Caenorhabditis elegans homolog of human heterogeneous nuclear ribonucleoproteins Q and R, binds to UCUAUC in the context of unc-52 intronic regulatory sequences as well as to RNAs containing tandem repeats of this sequence. The three Us in the hexamer are the most important determinants of this binding specificity. We demonstrate, using RNA interference, that HRP-2 regulates the alternative splicing of two genes, unc-52 and lin-10, both of which have cassette exons flanked by an intronic UCUAUC motif. We propose that HRP-2 is a protein responsible for regulating alternative splicing through binding interactions with the UCUAUC sequence.

  17. Analyzing alternative splicing data of splice junction arrays from Parkinson patients' leukocytes before and after deep brain stimulation as compared with control donors

    Directory of Open Access Journals (Sweden)

    Lilach Soreq

    2015-09-01

    Full Text Available Few studies so far examined alternative splicing alterations in blood cells of neurodegenerative disease patients, particularly Parkinson's disease (PD. Prototype junction microarrays interrogate known human genome junctions and enable characterization of alternative splicing events; however, the analysis is not straightforward and different methods can be used to estimate junction-specific alternative splicing events (some of which can also be applied for analyzing RNA sequencing junction-level data. In this study, we characterized alternative splicing changes in blood leukocyte samples from Parkinson's patients prior to, and following deep brain stimulation (DBS treatment; both on stimulation and following 1 h off electrical stimulation. Here, we describe in detail analysis approaches for junction microarrays and provide suggestions for further analyses to delineate transcript level effects of the observed alterations as well as detection of microRNA binding sites and protein domains in the alternatively spliced target regions spanning across both untranslated and the coding regions of the targets. The raw expression data files are publically available in the Gene Expression Omnibus (GEO database (accession number: GSE37591 and in Synapse, and can be re-analyzed. The results may be useful for designing of future experiments and cross correlations with other datasets from PD or patients having other neurodegenerative diseases.

  18. Depolarization-mediated regulation of alternative splicing

    Directory of Open Access Journals (Sweden)

    Alok eSharma

    2011-12-01

    Full Text Available Alternative splicing in eukaryotes plays an important role in regulating gene expression by selectively including alternative exons. A wealth of information has been accumulated that explains how alternative exons are selected in a developmental stage- or tissue-specific fashion. However, our knowledge of how cells respond to environmental changes to alter alternative splicing is very limited. For example, although a number of alternative exons have been shown to be regulated by calcium level alterations, the underlying mechanisms are not well understood. As calcium signaling in neurons plays a crucial role in essential neuronal functions such as learning and memory formation, it is important to understand how this process is regulated at every level in gene expression. The significance of the dynamic control of alternative splicing in response to changes of calcium levels has been largely unappreciated. In this communication, we will summarize the recent advances in calcium signaling-mediated alternative splicing that have provided some insights into the important regulatory mechanisms. In addition to describing the cis-acting RNA elements on the pre-mRNA molecules that respond to changes of intracellular calcium levels, we will summarize how splicing regulators change and affect alternative splicing in this process. We will also discuss a novel mode of calcium-mediated splicing regulation at the level of chromatin structure and transcription.

  19. U2 toggles iteratively between the stem IIa and stem IIc conformations to promote pre-mRNA splicing.

    Science.gov (United States)

    Hilliker, Angela K; Mefford, Melissa A; Staley, Jonathan P

    2007-04-01

    To ligate exons in pre-messenger RNA (pre-mRNA) splicing, the spliceosome must reposition the substrate after cleaving the 5' splice site. Because spliceosomal small nuclear RNAs (snRNAs) bind the substrate, snRNA structures may rearrange to reposition the substrate. However, such rearrangements have remained undefined. Although U2 stem IIc inhibits binding of U2 snRNP to pre-mRNA during assembly, we found that weakening U2 stem IIc suppressed a mutation in prp16, a DExD/H box ATPase that promotes splicing after 5' splice site cleavage. The prp16 mutation was also suppressed by mutations flanking stem IIc, suggesting that Prp16p facilitates a switch from stem IIc to the mutually exclusive U2 stem IIa, which activates binding of U2 to pre-mRNA during assembly. Providing evidence that stem IIa switches back to stem IIc before exon ligation, disrupting stem IIa suppressed 3' splice site mutations, and disrupting stem IIc impaired exon ligation. Disrupting stem IIc also exacerbated the 5' splice site cleavage defects of certain substrate mutations, suggesting a parallel role for stem IIc at both catalytic stages. We propose that U2, much like the ribosome, toggles between two conformations--a closed stem IIc conformation that promotes catalysis and an open stem IIa conformation that promotes substrate binding and release.

  20. An Alternate Splicing Variant of the Human Telomerase Catalytic Subunit Inhibits Telomerase Activity

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    Xiaoming Yi

    2000-09-01

    Full Text Available Telomerase, a cellular reverse transcriptase, adds telomeric repeats to chromosome ends. In normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to proliferative senescence. Introduction of the telomerase (hTERT cDNA is sufficient to produce telomerase activity and immortalize normal human cells, suggesting that the repression of telomerase activity is transcriptional. The telomerase transcript has been shown to have at least six alternate splicing sites (four insertion sites and two deletion sites, and variants containing both or either of the deletion sites are present during development and in a panel of cancer cell lines we surveyed. One deletion (β site and all four insertions cause premature translation terminations, whereas the other deletion (α site is 36 by and lies within reverse transcriptase (RT motif A, suggesting that this deletion variant may be a candidate as a dominant-negative inhibitor of telomerase. We have cloned three alternately spliced hTERT variants that contain the α,β or both α and,β deletion sites. These alternate splicing variants along with empty vector and wild-type hTERT were introduced into normal human fibroblasts and several telomerase-positive immortal and tumor cell lines. Expression of the α site deletion variant (hTERT α− construct was confirmed by Western blotting. We found that none of the three alternate splicing variants reconstitutes telomerase activity in fibroblasts. However, hTERT α− inhibits telomerase activities in telomerase-positive cells, causes telomere shortening and eventually cell death. This alternately spliced dominant-negative variant may be important in understanding telomerase regulation during development, differentiation and in cancer progression.

  1. Skipping of exons by premature termination of transcription and alternative splicing within intron-5 of the sheep SCF gene: a novel splice variant.

    Directory of Open Access Journals (Sweden)

    Siva Arumugam Saravanaperumal

    Full Text Available Stem cell factor (SCF is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM, SCF is produced either as a membrane-bound (- or soluble (+ forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR. Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp cDNA encodes a precursor protein of 274 amino acids (aa, commonly known as 'soluble' isoform. In contrast, the shorter (835 and/or 725 bp cDNA was found to be a 'novel' mRNA splice variant. It contains an open reading frame (ORF corresponding to a truncated protein of 181 aa (vs 245 aa with an unique C-terminus lacking the primary proteolytic segment (28 aa right after the D(175G site which is necessary to produce 'soluble' form of SCF. This alternative splice (AS variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5-exon 6 (948 bp with a premature termination codon (PTC whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6-9/10. We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+ and/or absence (- of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals.

  2. Splicing pattern - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us ASTRA Splicing pattern Data detail Data name Splicing pattern DOI 10.18908/lsdba.nbdc00371-0...04 Description of data contents The patterns of alternative splicing/transcriptional initiation Data file Fi...le name: astra_splicing_pattern.zip File URL: ftp://ftp.biosciencedbc.jp/archive/astra/LATEST/astra_splicing_pat...ogodb/view/astra_splicing_pattern#en Data acquisition method For the five organisms (H. sapiens, M. musculus...apping data into bit arrays, detection of splicing patterns and distribution to t

  3. Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system.

    Science.gov (United States)

    Ohrt, Thomas; Odenwälder, Peter; Dannenberg, Julia; Prior, Mira; Warkocki, Zbigniew; Schmitzová, Jana; Karaduman, Ramazan; Gregor, Ingo; Enderlein, Jörg; Fabrizio, Patrizia; Lührmann, Reinhard

    2013-07-01

    Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5' and 3' exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3' splice site (3'SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3'SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing.

  4. Topography as a driver of cryptic speciation in the high-elevation cape sedge Tetraria triangularis (Boeck.) C. B. Clarke (Cyperaceae: Schoeneae).

    Science.gov (United States)

    Britton, Matthew N; Hedderson, Terry A; Anthony Verboom, G

    2014-08-01

    Since some speciation mechanisms are more likely to generate morphological disparity than others, the general failure of vascular plant taxonomists to recognize cryptic diversity may bias perceptions about speciation process in plants. While the exceptional floristic richness of the South African Cape has largely been attributed to adaptive divergence ('ecological' speciation), a combination of climatic dynamism and complex topography has likely provided ample opportunities for 'non-ecological' vicariant speciation, a mechanism which is perhaps more likely to produce cryptic species. We explore the role of topography as a driver of 'non-ecological' speciation in the high-elevation sedge Tetraria triangularis. Within this species, molecular and morphological data reveal five cryptic or semi-cryptic lineages of Miocene-Pliocene age which qualify as evolutionary species. At least three of these maintain their distinctness in sites of sympatry, identifying them as biological species. Negligible range overlap, and the identification of topography as a significant predictor of range turnover, identifies speciation as allopatric and a result of impeded gene flow across low-elevation topographic features. Weak morphological and ecological divergence implies a limited role for adaptive divergence in powering speciation, with character displacement in sympatry possibly arising as a consequence of interspecific competition. Although we cannot exclude a role for disruptive selection in species differentiation, we identify isolation of populations on topographically separated mountains as the principal motor of speciation. We suggest that the importance of topography in the genesis of Cape floristic diversity has been inadequately acknowledged.

  5. Anti-tumor activity of splice-switching oligonucleotides

    OpenAIRE

    Bauman, John A; Li, Shyh-Dar; Yang, Angela; Huang, Leaf; Kole, Ryszard

    2010-01-01

    Alternative splicing has emerged as an important target for molecular therapies. Splice-switching oligonucleotides (SSOs) modulate alternative splicing by hybridizing to pre-mRNA sequences involved in splicing and blocking access to the transcript by splicing factors. Recently, the efficacy of SSOs has been established in various animal disease models; however, the application of SSOs against cancer targets has been hindered by poor in vivo delivery of antisense therapeutics to tumor cells. T...

  6. CTCF:from insulators to alternative splicing regulation

    Institute of Scientific and Technical Information of China (English)

    Alberto R Kornblihtt

    2012-01-01

    The zinc-finger DNA-binding protein CTCF has been known for being a constituent of insulators.A recent paper in Nature reports an unforeseen intragenic role for CTCF that links DNA methylation with alternative splicing.By binding to its target DNA site placed within an alternative exon,CTCF creates a roadblock to transcriptional elongation that favors inclusion of the exon into mature mRNA.DNA methylation prevents CTCF binding,which releases pol Ⅱ transient blockage and promotes exon exclusion.

  7. Faster exon assembly by sparse spliced alignment

    CERN Document Server

    Tiskin, Alexander

    2007-01-01

    Assembling a gene from candidate exons is an important problem in computational biology. Among the most successful approaches to this problem is \\emph{spliced alignment}, proposed by Gelfand et al., which scores different candidate exon chains within a DNA sequence of length $m$ by comparing them to a known related gene sequence of length n, $m = \\Theta(n)$. Gelfand et al.\\ gave an algorithm for spliced alignment running in time O(n^3). Kent et al.\\ considered sparse spliced alignment, where the number of candidate exons is O(n), and proposed an algorithm for this problem running in time O(n^{2.5}). We improve on this result, by proposing an algorithm for sparse spliced alignment running in time O(n^{2.25}). Our approach is based on a new framework of \\emph{quasi-local string comparison}.

  8. Protein splicing and its evolution in eukaryotes

    Directory of Open Access Journals (Sweden)

    Starokadomskyy P. L.

    2010-02-01

    Full Text Available Inteins, or protein introns, are parts of protein sequences that are post-translationally excised, their flanking regions (exteins being spliced together. This process was called protein splicing. Originally inteins were found in prokaryotic or unicellular eukaryotic organisms. But the general principles of post-translation protein rearrangement are evolving yielding different post-translation modification of proteins in multicellular organisms. For clarity, these non-intein mediated events call either protein rearrangements or protein editing. The most intriguing example of protein editing is proteasome-mediated splicing of antigens in vertebrates that may play important role in antigen presentation. Other examples of protein rearrangements are maturation of Hg-proteins (critical receptors in embryogenesis as well as maturation of several metabolic enzymes. Despite a lack of experimental data we try to analyze some intriguing examples of protein splicing evolution.

  9. Alcoholism and Alternative Splicing of Candidate Genes

    OpenAIRE

    Toshikazu Sasabe; Shoichi Ishiura

    2010-01-01

    Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor) may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports sugg...

  10. Targeting RNA-splicing for SMA treatment.

    Science.gov (United States)

    Zhou, Jianhua; Zheng, Xuexiu; Shen, Haihong

    2012-03-01

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

  11. Sperm storage mediated by cryptic female choice for nuptial gifts.

    Science.gov (United States)

    Albo, Maria J; Bilde, Trine; Uhl, Gabriele

    2013-12-07

    Polyandrous females are expected to discriminate among males through postcopulatory cryptic mate choice. Yet, there is surprisingly little unequivocal evidence for female-mediated cryptic sperm choice. In species in which nuptial gifts facilitate mating, females may gain indirect benefits through preferential storage of sperm from gift-giving males if the gift signals male quality. We tested this hypothesis in the spider Pisaura mirabilis by quantifying the number of sperm stored in response to copulation with males with or without a nuptial gift, while experimentally controlling copulation duration. We further assessed the effect of gift presence and copulation duration on egg-hatching success in matings with uninterrupted copulations with gift-giving males. We show that females mated to gift-giving males stored more sperm and experienced 17% higher egg-hatching success, compared with those mated to no-gift males, despite matched copulation durations. Uninterrupted copulations resulted in both increased sperm storage and egg-hatching success. Our study confirms the prediction that the nuptial gift as a male signal is under positive sexual selection by females through cryptic sperm storage. In addition, the gift facilitates longer copulations and increased sperm transfer providing two different types of advantage to gift-giving in males.

  12. Cryptic individual scaling relationships and the evolution of morphological scaling.

    Science.gov (United States)

    Dreyer, Austin P; Saleh Ziabari, Omid; Swanson, Eli M; Chawla, Akshita; Frankino, W Anthony; Shingleton, Alexander W

    2016-08-01

    Morphological scaling relationships between organ and body size-also known as allometries-describe the shape of a species, and the evolution of such scaling relationships is central to the generation of morphological diversity. Despite extensive modeling and empirical tests, however, the modes of selection that generate changes in scaling remain largely unknown. Here, we mathematically model the evolution of the group-level scaling as an emergent property of individual-level variation in the developmental mechanisms that regulate trait and body size. We show that these mechanisms generate a "cryptic individual scaling relationship" unique to each genotype in a population, which determines body and trait size expressed by each individual, depending on developmental nutrition. We find that populations may have identical population-level allometries but very different underlying patterns of cryptic individual scaling relationships. Consequently, two populations with apparently the same morphological scaling relationship may respond very differently to the same form of selection. By focusing on the developmental mechanisms that regulate trait size and the patterns of cryptic individual scaling relationships they produce, our approach reveals the forms of selection that should be most effective in altering morphological scaling, and directs researcher attention on the actual, hitherto overlooked, targets of selection.

  13. Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.

    Science.gov (United States)

    Zhang, Jian; Lieu, Yen K; Ali, Abdullah M; Penson, Alex; Reggio, Kathryn S; Rabadan, Raul; Raza, Azra; Mukherjee, Siddhartha; Manley, James L

    2015-08-25

    Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.

  14. Cryptic extinction of a common Pacific lizard Emoia impar (Squamata, Scincidae) from the Hawaiian Islands.

    Science.gov (United States)

    Fisher, Robert; Ineich, Ivan

    2012-01-01

    Most documented declines of tropical reptiles are of dramatic or enigmatic species. Declines of widespread species tend to be cryptic. The early (1900s) decline and extinction of the common Pacific skink Emoia impar from the Hawaiian Islands is documented here through an assessment of literature, museum vouchers and recent fieldwork. This decline appears contemporaneous with the documented declines of invertebrates and birds across the Hawaiian Islands. A review of the plausible causal factors indicates that the spread of the introduced big-headed ant Pheidole megacephala is the most likely factor in this lizard decline. The introduction and spread of a similar skink Lampropholis delicata across the islands appears to temporally follow the decline of E. impar, although there is no evidence of competition between these species. It appears that L. delicata is spreading to occupy the niche vacated by the extirpated E. impar. Further confusion exists because the skink E. cyanura, which is very similar in appearance to E. impar, appears to have been introduced to one site within a hotel on Kaua'i and persisted as a population at that site for approximately 2 decades (1970s–1990s) but is now also extirpated. This study highlights the cryptic nature of this early species extinction as evidence that current biogeographical patterns of non-charismatic or enigmatic reptiles across the Pacific may be the historical result of early widespread invasion by ants. Conservation and restoration activities for reptiles in the tropical Pacific should consider this possibility and evaluate all evidence prior to any implementation.

  15. Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene

    Institute of Scientific and Technical Information of China (English)

    Guang-Jing Hu; Jia Chen; Xiao-Nan Zhao; Jia-Jia Xu; Dong-Qing Guo; Ming Lu; Ming Zhu

    2013-01-01

    Trans-splicing,a process involving the cleavage and joining of two separate transcripts,can expand the transcriptome and proteome in eukaryotes.Chimeric RNAs generated by trans-splicing are increasingly described in literatures.The widespread presence of antibiotic resistance genes in natural environments and human intestines is becoming an important challenge for public health.Certain antibiotic resistance genes,such as ampicillin resistance gene (Amp),are frequently used in recombinant plasmids.Until now,trans-splicing involving recombinant plasmid-derived exogenous transcripts and endogenous cellular RNAs has not been reported.Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) is a key enzyme involved in cellular cholesterol homeostasis.The 4.3-kb human ACAT1 chimeric mRNA can produce 50-kDa and 56-kDa isoforms with different enzymatic activities.Here,we show that human ACAT1 56-kDa isoform is produced from an mRNA species generated through the trans-splicing of an exogenous transcript encoded by the antisense strand of Ampr (asAmp) present in common Ampr-plasmids and the 4.3-kb endogenous ACAT1 chimeric mRNA,which is presumably processed through a prior event of interchromosomal trans-splicing.Strikingly,DNA fragments containing the asAmp with an upstream recombined cryptic promoter and the corresponding exogenous asAmp transcripts have been detected in human cells.Our findings shed lights on the mechanism of human ACAT1 56-kDa isoform production,reveal an exogenous-endogenous trans-splicing system,in which recombinant plasmid-derived exogenous transcripts are linked with endogenous cellular RNAs in human cells,and suggest that exogenous DNA might affect human gene expression at both DNA and RNA levels.

  16. Splice variants of Na(V1.7 sodium channels have distinct β subunit-dependent biophysical properties.

    Directory of Open Access Journals (Sweden)

    Clare Farmer

    Full Text Available Genes encoding the α subunits of neuronal sodium channels have evolutionarily conserved sites of alternative splicing but no functional differences have been attributed to the splice variants. Here, using Na(V1.7 as an exemplar, we show that the sodium channel isoforms are functionally distinct when co-expressed with β subunits. The gene, SCN9A, encodes the α subunit of the Na(V1.7 channel, and contains both sites of alternative splicing that are highly conserved. In conditions where the intrinsic properties of the Na(V1.7 splice variants were similar when expressed alone, co-expression of β1 subunits had different effects on channel availability that were determined by splicing at either site in the α subunit. While the identity of exon 5 determined the degree to which β1 subunits altered voltage-dependence of activation (P = 0.027, the length of exon 11 regulated how far β1 subunits depolarised voltage-dependence of inactivation (P = 0.00012. The results could have a significant impact on channel availability, for example with the long version of exon 11, the co-expression of β1 subunits could lead to nearly twice as large an increase in channel availability compared to channels containing the short version. Our data suggest that splicing can change the way that Na(V channels interact with β subunits. Because splicing is conserved, its unexpected role in regulating the functional impact of β subunits may apply to multiple voltage-gated sodium channels, and the full repertoire of β subunit function may depend on splicing in α subunits.

  17. Negative and Positive mRNA Splicing Elements Act Competitively To Regulate Human Immunodeficiency Virus Type 1 Vif Gene Expression▿

    Science.gov (United States)

    Exline, C. M.; Feng, Z.; Stoltzfus, C. M.

    2008-01-01

    Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3′ splice sites (3′ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3′ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5′ splice site (5′ss) downstream of exon 2 (5′ss D2). Here we show that the mutations within 5′ss D2 that are predicted to lower or increase the affinity of the 5′ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5′ss D2 was not necessary for the effect of 5′ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5′ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5′-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5′ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication. PMID:18272582

  18. A pan-cancer analysis of transcriptome changes associated with somatic mutations in U2AF1 reveals commonly altered splicing events.

    Directory of Open Access Journals (Sweden)

    Angela N Brooks

    Full Text Available Although recurrent somatic mutations in the splicing factor U2AF1 (also known as U2AF35 have been identified in multiple cancer types, the effects of these mutations on the cancer transcriptome have yet to be fully elucidated. Here, we identified splicing alterations associated with U2AF1 mutations across distinct cancers using DNA and RNA sequencing data from The Cancer Genome Atlas (TCGA. Using RNA-Seq data from 182 lung adenocarcinomas and 167 acute myeloid leukemias (AML, in which U2AF1 is somatically mutated in 3-4% of cases, we identified 131 and 369 splicing alterations, respectively, that were significantly associated with U2AF1 mutation. Of these, 30 splicing alterations were statistically significant in both lung adenocarcinoma and AML, including three genes in the Cancer Gene Census, CTNNB1, CHCHD7, and PICALM. Cell line experiments expressing U2AF1 S34F in HeLa cells and in 293T cells provide further support that these altered splicing events are caused by U2AF1 mutation. Consistent with the function of U2AF1 in 3' splice site recognition, we found that S34F/Y mutations cause preferences for CAG over UAG 3' splice site sequences. This report demonstrates consistent effects of U2AF1 mutation on splicing in distinct cancer cell types.

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

    Science.gov (United States)

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

    2014-02-10

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

  20. Splicing of a C. elegans myosin pre-mRNA in a human nuclear extract

    Energy Technology Data Exchange (ETDEWEB)

    Ogg, S.C.; Anderson, P.; Wickens, M.P. (Univ. of Wisconsin, Madison (USA))

    1990-01-11

    Splicing of mammalian introns requires that the intron possess at least 80 nucleotides. This length requirement presumably reflects the constraints of accommodating multiple snRNPs simultaneously in the same intro. In the free-living nematode, C. elegans, introns typically are 45 to 55 nucleotides in length. In this report, the authors determine whether C. elegans introns can obviate the mammalian length requirement by virtue of their structure or sequence. They demonstrate that a 53 nucleotide intron from the unc-54 gene of C. elegans does not undergo splicing in a mammalian (HeLa) nuclear extract. However, insertion of 31 nucleotides of foreign, prokaryotic sequence into the same intron results in efficient splicing. The observed splicing proceeds by the same two-step mechanism observed with mammalian introns, and exploits the same 3{prime} and 5{prime} sites as are used in C. elegans. The branch point used lies in the inserted sequences. They conclude that C. elegans splicing components are either fewer in number or smaller than their mammalian counterparts.

  1. Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins.

    Science.gov (United States)

    Huelga, Stephanie C; Vu, Anthony Q; Arnold, Justin D; Liang, Tiffany Y; Liu, Patrick P; Yan, Bernice Y; Donohue, John Paul; Shiue, Lily; Hoon, Shawn; Brenner, Sydney; Ares, Manuel; Yeo, Gene W

    2012-02-23

    Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here, we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and autoregulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.

  2. Integrative Genome-wide Analysis Reveals Cooperative Regulation of Alternative Splicing by hnRNP Proteins

    Directory of Open Access Journals (Sweden)

    Stephanie C. Huelga

    2012-02-01

    Full Text Available Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here, we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS in human cells. Using splicing-sensitive microarrays, crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and autoregulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.

  3. Global impact of RNA splicing on transcriptome remodeling in the heart

    Institute of Scientific and Technical Information of China (English)

    Chen GAO; Yibin WANG

    2012-01-01

    In the eukaryotic transcriptome,both the numbers of genes and different RNA species produced by each gene contribute to the overall complexity.These RNA species are generated by the utilization of different transcriptional initiation or termination sites,or more commonly,from different messenger RNA (mRNA) splicing events.Among the 30 000+ genes in human genome,it is estimated that more than 95% of them can generate more than one gene product via alternative RNA splicing.The protein products generated from different RNA splicing variants can have different intracellular localization,activity,or tissue-distribution.Therefore,alternative RNA splicing is an important molecular process that contributes to the overall complexity of the genome and the functional specificity and diversity among different cell types.In this review,we will discuss current efforts to unravel the full complexity of the cardiac transcriptome using a deep-sequencing approach,and highlight the potential of this technology to uncover the global impact of RNA splicing on the transcriptome during development and diseases of the heart.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

  6. Global impact of RNA splicing on transcriptome remodeling in the heart.

    Science.gov (United States)

    Gao, Chen; Wang, Yibin

    2012-08-01

    In the eukaryotic transcriptome, both the numbers of genes and different RNA species produced by each gene contribute to the overall complexity. These RNA species are generated by the utilization of different transcriptional initiation or termination sites, or more commonly, from different messenger RNA (mRNA) splicing events. Among the 30,000+ genes in human genome, it is estimated that more than 95% of them can generate more than one gene product via alternative RNA splicing. The protein products generated from different RNA splicing variants can have different intracellular localization, activity, or tissue-distribution. Therefore, alternative RNA splicing is an important molecular process that contributes to the overall complexity of the genome and the functional specificity and diversity among different cell types. In this review, we will discuss current efforts to unravel the full complexity of the cardiac transcriptome using a deep-sequencing approach, and highlight the potential of this technology to uncover the global impact of RNA splicing on the transcriptome during development and diseases of the heart.

  7. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani.

    Science.gov (United States)

    McNeil, Bonnie A; Simon, Dawn M; Zimmerly, Steven

    2014-02-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5' splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5' exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns.

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

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    Junyu Zhang

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

  9. Differential splicing using whole-transcript microarrays

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    Robinson Mark D

    2009-05-01

    Full Text Available Abstract Background The latest generation of Affymetrix microarrays are designed to interrogate expression over the entire length of every locus, thus giving the opportunity to study alternative splicing genome-wide. The Exon 1.0 ST (sense target platform, with versions for Human, Mouse and Rat, is designed primarily to probe every known or predicted exon. The smaller Gene 1.0 ST array is designed as an expression microarray but still interrogates expression with probes along the full length of each well-characterized transcript. We explore the possibility of using the Gene 1.0 ST platform to identify differential splicing events. Results We propose a strategy to score differential splicing by using the auxiliary information from fitting the statistical model, RMA (robust multichip analysis. RMA partitions the probe-level data into probe effects and expression levels, operating robustly so that if a small number of probes behave differently than the rest, they are downweighted in the fitting step. We argue that adjacent poorly fitting probes for a given sample can be evidence of differential splicing and have designed a statistic to search for this behaviour. Using a public tissue panel dataset, we show many examples of tissue-specific alternative splicing. Furthermore, we show that evidence for putative alternative splicing has a strong correspondence between the Gene 1.0 ST and Exon 1.0 ST platforms. Conclusion We propose a new approach, FIRMAGene, to search for differentially spliced genes using the Gene 1.0 ST platform. Such an analysis complements the search for differential expression. We validate the method by illustrating several known examples and we note some of the challenges in interpreting the probe-level data. Software implementing our methods is freely available as an R package.

  10. Vitamin D and alternative splicing of RNA.

    Science.gov (United States)

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

    2015-04-01

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

  11. Coding potential of the products of alternative splicing in human.

    KAUST Repository

    Leoni, Guido

    2011-01-20

    BACKGROUND: Analysis of the human genome has revealed that as much as an order of magnitude more of the genomic sequence is transcribed than accounted for by the predicted and characterized genes. A number of these transcripts are alternatively spliced forms of known protein coding genes; however, it is becoming clear that many of them do not necessarily correspond to a functional protein. RESULTS: In this study we analyze alternative splicing isoforms of human gene products that are unambiguously identified by mass spectrometry and compare their properties with those of isoforms of the same genes for which no peptide was found in publicly available mass spectrometry datasets. We analyze them in detail for the presence of uninterrupted functional domains, active sites as well as the plausibility of their predicted structure. We report how well each of these strategies and their combination can correctly identify translated isoforms and derive a lower limit for their specificity, that is, their ability to correctly identify non-translated products. CONCLUSIONS: The most effective strategy for correctly identifying translated products relies on the conservation of active sites, but it can only be applied to a small fraction of isoforms, while a reasonably high coverage, sensitivity and specificity can be achieved by analyzing the presence of non-truncated functional domains. Combining the latter with an assessment of the plausibility of the modeled structure of the isoform increases both coverage and specificity with a moderate cost in terms of sensitivity.

  12. A biophysical model for identifying splicing regulatory elements and their interactions.

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    Ji Wen

    Full Text Available Alternative splicing (AS of precursor mRNA (pre-mRNA is a crucial step in the expression of most eukaryotic genes. Splicing factors (SFs play an important role in AS regulation by binding to the cis-regulatory elements on the pre-mRNA. Although many splicing factors (SFs and their binding sites have been identified, their combinatorial regulatory effects remain to be elucidated. In this paper, we derive a biophysical model for AS regulation that integrates combinatorial signals of cis-acting splicing regulatory elements (SREs and their interactions. We also develop a systematic framework for model inference. Applying the biophysical model to a human RNA-Seq data set, we demonstrate that our model can explain 49.1%-66.5% variance of the data, which is comparable to the best result achieved by biophysical models for transcription. In total, we identified 119 SRE pairs between different regions of cassette exons that may regulate exon or intron definition in splicing, and 77 SRE pairs from the same region that may arise from a long motif or two different SREs bound by different SFs. Particularly, putative binding sites of polypyrimidine tract-binding protein (PTB, heterogeneous nuclear ribonucleoprotein (hnRNP F/H and E/K are identified as interacting SRE pairs, and have been shown to be consistent with the interaction models proposed in previous experimental results. These results show that our biophysical model and inference method provide a means of quantitative modeling of splicing regulation and is a useful tool for identifying SREs and their interactions. The software package for model inference is available under an open source license.

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

    Science.gov (United States)

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

    2014-03-01

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

  14. Cryptic mosaicism involving a second chromosome X in patients with Turner syndrome

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    A. Araújo

    2008-05-01

    Full Text Available The high abortion rate of 45,X embryos indicates that patients with Turner syndrome and 45,X karyotype could be mosaics, in at least one phase of embryo development or cellular lineage, due to the need for the other sex chromosome presence for conceptus to be compatible with life. In cases of structural chromosomal aberrations or hidden mosaicism, conventional cytogenetic techniques can be ineffective and molecular investigation is indicated. Two hundred and fifty patients with Turner syndrome stigmata were studied and 36 who had female genitalia and had been cytogenetically diagnosed as having "pure" 45,X karyotype were selected after 100 metaphases were analyzed in order to exclude mosaicism and the presence of genomic Y-specific sequences (SRY, TSPY, and DAZ was excluded by PCR. Genomic DNA was extracted from peripheral blood and screened by the human androgen receptor (HUMARA assay. The HUMARA gene has a polymorphic CAG repeat and, in the presence of a second chromosome with a different HUMARA allele, a second band will be amplified by PCR. Additionally, the CAG repeats contain two methylation-sensitive HpaII enzyme restriction sites, which can be used to verify skewed inactivation. Twenty-five percent (9/36 of the cases showed a cryptic mosaicism involving a second X and approximately 14% (5/36, or 55% (5/9 of the patients with cryptic mosaicism, also presented skewed inactivation. The laboratory identification of the second X chromosome and its inactivation pattern are important for the clinical management (hormone replacement therapy, and inclusion in an oocyte donation program and prognostic counseling of patients with Turner syndrome.

  15. Cryptic lineages and diversification of an endemic anole lizard (Squamata, Dactyloidae) of the Cerrado hotspot.

    Science.gov (United States)

    Guarnizo, Carlos E; Werneck, Fernanda P; Giugliano, Lilian G; Santos, Marcella G; Fenker, Jéssica; Sousa, Lucas; D'Angiolella, Annelise B; Dos Santos, Adriana R; Strüssmann, Christine; Rodrigues, Miguel T; Dorado-Rodrigues, Tainá F; Gamble, Tony; Colli, Guarino R

    2016-01-01

    The Cerrado is a wide Neotropical savanna with tremendously high endemic diversity. Yet, it is not clear what the prevalent processes leading to such diversification are. We used the Cerrado-endemic lizard Norops meridionalis to investigate the main abiotic factors that promoted genetic divergence, the timings of these divergence events, and how these relate to cryptic diversity in the group. We sequenced mitochondrial and nuclear genes from 21 sites of N. meridionalis to generate species tree, divergence time estimations, and estimate species limits. We also performed population-level analysis and estimated distribution models to test the roles of niche conservatism and divergence in the group diversification. We found that N. meridionalis is composed by at least five cryptic species. Divergence time estimations suggest that the deepest branches split back into the early-mid Miocene, when most of the geophysical activity of the Cerrado took place. The deep divergences found in N. meridionalis suggest that beta anoles invaded South America much earlier than previously thought. Recent published evidence supports this view, indicating that the Panama gap closed as early as 15 mya, allowing for an early invasion of Norops into South America. The spatial pattern of diversification within N. meridionalis follows a northwest-southeast direction, which is consistent across several species of vertebrates endemic to the Cerrado. Also, we found evidence for non-stationary isolation by distance, which occurs when genetic differentiation depends on space. Our preliminary data in two out of five lineages suggest that niche conservatism is an important mechanism that promoted geographic fragmentation in the group.

  16. Deep mtDNA divergences indicate cryptic species in a fig-pollinating wasp

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    Martin Joanne

    2006-10-01

    Full Text Available Abstract Background Figs and fig-pollinating wasps are obligate mutualists that have coevolved for ca 90 million years. They have radiated together, but do not show strict cospeciation. In particular, it is now clear that many fig species host two wasp species, so there is more wasp speciation than fig speciation. However, little is known about how fig wasps speciate. Results We studied variation in 71 fig-pollinating wasps from across the large geographic range of Ficus rubiginosa in Australia. All wasps sampled belong to one morphological species (Pleistodontes imperialis, but we found four deep mtDNA clades that differed from each other by 9–17% nucleotides. As these genetic distances exceed those normally found within species and overlap those (10–26% found between morphologically distinct Pleistodontes species, they strongly suggest cryptic fig wasp species. mtDNA clade diversity declines from all four present in Northern Queensland to just one in Sydney, near the southern range limit. However, at most sites multiple clades coexist and can be found in the same tree or even the same fig fruit and there is no evidence for parallel sub-division of the host fig species. Both mtDNA data and sequences from two nuclear genes support the monophyly of the "P. imperialis complex" relative to other Pleistodontes species, suggesting that fig wasp divergence has occurred without any host plant shift. Wasps in clade 3 were infected by a single strain (W1 of Wolbachia bacteria, while those in other clades carried a double infection (W2+W3 of two other strains. Conclusion Our study indicates that cryptic fig-pollinating wasp species have developed on a single host plant species, without the involvement of host plant shifts, or parallel host plant divergence. Despite extensive evidence for coevolution between figs and fig wasps, wasp speciation may not always be linked strongly with fig speciation.

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

    Science.gov (United States)

    McNeil, Bonnie A; Zimmerly, Steven

    2014-06-01

    Group II introns are ribozymes in bacterial and organellar genomes that function as self-splicing introns and as retroelements. Previously, we reported that the group II intron C.te.I1 of Clostridium tetani alternatively splices in vivo to produce five distinct coding mRNAs. Accurate fusion of upstream and downstream reading frames requires a shifted 5' splice site located 8 nt upstream of the usual 5' GUGYG motif. This site is specified by the ribozyme through an altered intron/exon-binding site 1 (IBS1-EBS1) pairing. Here we use mutagenesis and self-splicing assays to investigate in more detail the significance of the structural features of the C.te.I1 ribozyme. The shifted 5' splice site is shown to be affected by structures in addition to IBS1-EBS1, and unlike other group II introns, C.te.I1 appears to require a spacer between IBS1 and the GUGYG motif. In addition, the mechanism of 3' exon recognition is modified from the ancestral IIB mechanism to a IIA-like mechanism that appears to be longer than the typical single base-pair interaction and may extend up to 4 bp. The novel ribozyme properties that have evolved for C.te.I1 illustrate the plasticity of group II introns in adapting new structural and catalytic properties that can be utilized to affect gene expression. © 2014 McNeil and Zimmerly; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  18. RPL30 regulation of splicing reveals distinct roles for Cbp80 in U1 and U2 snRNP cotranscriptional recruitment.

    Science.gov (United States)

    Bragulat, Mireia; Meyer, Markus; Macías, Sara; Camats, Maria; Labrador, Mireia; Vilardell, Josep

    2010-10-01

    Pre-mRNA splicing is catalyzed by the spliceosome, and its control is essential for correct gene expression. While splicing repressors typically interfere with transcript recognition by spliceosomal components, the yeast protein L30 blocks spliceosomal rearrangements required for the engagement of U2 snRNP (small ribonucleoprotein particle) to its own transcript RPL30. Using a mutation in the RPL30 binding site that disrupts this repression, we have taken a genetic approach to reveal that regulation of splicing is restored in this mutant by deletion of the cap-binding complex (CBC) component Cbp80. Indeed, our data indicate that Cbp80 plays distinct roles in the recognition of the intron by U1 and U2 snRNP. It promotes the initial 5' splice site recognition by U1 and, independently, facilitates U2 recruitment, depending on sequences located in the vicinity of the 5' splice site. These results reveal a novel function for CBC in splicing and imply that these molecular events can be the target of a splicing regulator.

  19. Discovery of a mammalian splice variant of myostatin that stimulates myogenesis.

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    Ferenc Jeanplong

    Full Text Available Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P<0.05, which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a

  20. Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging.

    Science.gov (United States)

    Rodríguez, Sofía A; Grochová, Diana; McKenna, Tomás; Borate, Bhavesh; Trivedi, Niraj S; Erdos, Michael R; Eriksson, Maria

    2016-04-01

    Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome-wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild-type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3-3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P aging disease Hutchinson-Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild-type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known.

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

    Science.gov (United States)

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

    2010-07-01

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

  2. Classical estrogen receptors and ERα splice variants in the mouse.

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    Debra L Irsik

    Full Text Available Estrogens exert a variety of effects in both reproductive and non-reproductive tissues. With the discovery of ERα splice variants, prior assumptions concerning tissue-specific estrogen signaling need to be re-evaluated. Accordingly, we sought to determine the expression of the classical estrogen receptors and ERα splice variants across reproductive and non-reproductive tissues of male and female mice. Western blotting revealed that the full-length ERα66 was mainly present in female reproductive tissues but was also found in non-reproductive tissues at lower levels. ERα46 was most highly expressed in the heart of both sexes. ERα36 was highly expressed in the kidneys and liver of female mice but not in the kidneys of males. ERβ was most abundant in non-reproductive tissues and in the ovaries. Because the kidney has been reported to be the most estrogenic non-reproductive organ, we sought to elucidate ER renal expression and localization. Immunofluorescence studies revealed ERα66 in the vasculature and the glomerulus. It was also found in the brush border of the proximal tubule and in the cortical collecting duct of female mice. ERα36 was evident in mesangial cells and tubular epithelial cells of both sexes, as well as podocytes of females but not males. ERβ was found primarily in the podocytes in female mice but was also present in the mesangial cells in both sexes. Within the renal cortex, ERα46 and ERα36 were mainly located in the membrane fraction although they were also present in the cytosolic fraction. Given the variability of expression patterns demonstrated herein, identification of the specific estrogen receptors expressed in a tissue is necessary for interpreting estrogenic effects. As this study revealed expression of the ERα splice variants at multiple sites within the kidney, further studies are warranted in order to elucidate the contribution of these receptors to renal estrogen responsiveness.

  3. Regulation of mammalian pre-mRNA splicing

    Institute of Scientific and Technical Information of China (English)

    HUI JingYi

    2009-01-01

    In eukaryotes, most protein-coding genes contain introns which are removed by precursor messenger RNA (pre-mRNA) splicing. Alternative splicing is a process by which multiple messenger RNAs (mRNAs) are generated from a single pre-mRNA, resulting in functionally distinct proteins. Recent genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene expression. Mis-regulation of splicing causes a wide range of human diseases. This review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA splicing. It also discusses emerging directions in the field of alternative splicing.

  4. Regulation of mammalian pre-mRNA splicing

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    In eukaryotes,most protein-coding genes contain introns which are removed by precursor messenger RNA(pre-mRNA) splicing.Alternative splicing is a process by which multiple messenger RNAs(mRNAs) are generated from a single pre-mRNA,resulting in functionally distinct proteins.Recent genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene expression.Mis-regulation of splicing causes a wide range of human diseases.This review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA splicing.It also discusses emerging directions in the field of alternative splicing.

  5. A study of alternative splicing in the pig

    DEFF Research Database (Denmark)

    Hillig, Ann-Britt Nygaard; Cirera Salicio, Susanna; Gilchrist, Michael J.;

    2010-01-01

    BACKGROUND: Since at least half of the genes in mammalian genomes are subjected to alternative splicing, alternative pre-mRNA splicing plays an important contribution to the complexity of the mammalian proteome. Expressed sequence tags (ESTs) provide evidence of a great number of possible...... and mouse, we find putative splice variants in about 30% of the contigs with more than 50 ESTs. Based on the criteria that a minimum of two EST sequences confirmed each splice event, a list of 100 genes with the most distinct tissue-specific alternative splice events was generated from the list...... of candidates. To confirm the tissue specificity of the splice events, 10 genes with functional annotation were randomly selected from which 16 individual splice events were chosen for experimental verification by quantitative PCR (qPCR). Six genes were shown to have tissue specific alternatively spliced...

  6. Widespread evolutionary conservation of alternatively spliced exons in caenorhabditis

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob L; Penny, David

    2007-01-01

    Alternative splicing (AS) contributes to increased transcriptome and proteome diversity in various eukaryotic lineages. Previous studies showed low levels of conservation of alternatively spliced (cassette) exons within mammals and within dipterans. We report a strikingly different pattern...

  7. High frequency of T cells specific for cryptic epitopes in melanoma patients

    DEFF Research Database (Denmark)

    Andersen, Rikke Sick; Andersen, Sofie Ramskov; Hjortsø, Mads Duus

    2013-01-01

    A number of cytotoxic T-cell epitopes are cryptic epitopes generated from non-conventional sources. These include epitopes that are encoded by alternative open reading frames or in generally non-coding genomic regions, such as introns. We have previously observed a frequent recognition of cryptic...

  8. Complete sequence of a cryptic virus from hemp (Cannabis sativa).

    Science.gov (United States)

    Ziegler, Angelika; Matoušek, Jaroslav; Steger, Gerhard; Schubert, Jörg

    2012-02-01

    Hemp (Cannabis sativa) was found to be a useful propagation host for hop latent virus, a carlavirus. However, when virus preparations were analysed by electron microscopy, along with the expected filamentous particles, spherical particles with a diameter of around 34 nm were found. RNA from virus preparations was purified, and cDNA was prepared and cloned. Sequence information was used to search databases, and the greatest similarity was found with Primula malacoides virus 1, a putative new member of the genus Partitivirus. The full sequences of RNA 1 and RNA 2 of this new hemp cryptic virus were obtained.

  9. Functional and evolutionary analysis of alternatively spliced genes is consistent with an early eukaryotic origin of alternative splicing

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Penny, David;

    2007-01-01

    Alternative splicing has been reported in various eukaryotic groups including plants, apicomplexans, diatoms, amoebae, animals and fungi. However, whether widespread alternative splicing has evolved independently in the different eukaryotic groups or was inherited from their last common ancestor...

  10. The implications of alternative splicing in the ENCODE protein complement

    DEFF Research Database (Denmark)

    Tress, Michael L.; Martelli, Pier Luigi; Frankish, Adam;

    2007-01-01

    Alternative premessenger RNA splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been...

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

    Science.gov (United States)

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

    2016-09-23

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

  12. 46 CFR 111.60-19 - Cable splices.

    Science.gov (United States)

    2010-10-01

    ... with section 25.11 of IEEE 45-2002 (incorporated by reference; see 46 CFR 110.10-1). ... 46 Shipping 4 2010-10-01 2010-10-01 false Cable splices. 111.60-19 Section 111.60-19 Shipping... REQUIREMENTS Wiring Materials and Methods § 111.60-19 Cable splices. (a) A cable must not be spliced in...

  13. 30 CFR 75.603 - Temporary splice of trailing cable.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Temporary splice of trailing cable. 75.603... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.603 Temporary splice of trailing cable. One temporary splice may be made in any trailing cable. Such trailing cable...

  14. Structural insights into RNA recognition by the alternative-splicing regulator muscleblind-like MBNL1

    Energy Technology Data Exchange (ETDEWEB)

    Teplova, Marianna; Patel, Dinshaw J. (MSKCC)

    2009-01-15

    Muscleblind-like (MBNL) proteins, regulators of developmentally programmed alternative splicing, harbor tandem CCCH zinc-finger (ZnF) domains that target pre-mRNAs containing YGCU(U/G)Y sequence elements (where Y is a pyrimidine). In myotonic dystrophy, reduced levels of MBNL proteins lead to aberrant alternative splicing of a subset of pre-mRNAs. The crystal structure of MBNL1 ZnF3/4 bound to r(CGCUGU) establishes that both ZnF3 and ZnF4 target GC steps, with site-specific recognition mediated by a network of hydrogen bonds formed primarily with main chain groups of the protein. The relative alignment of ZnF3 and ZnF4 domains is dictated by the topology of the interdomain linker, with a resulting antiparallel orientation of bound GC elements, supportive of a chain-reversal loop trajectory for MBNL1-bound pre-mRNA targets. We anticipate that MBNL1-mediated targeting of looped RNA segments proximal to splice-site junctions could contribute to pre-mRNA alternative-splicing regulation.

  15. Alternative splicing of the angiogenesis associated extra-domain B of fibronectin regulates the accessibility of the B-C loop of the type III repeat 8.

    Directory of Open Access Journals (Sweden)

    Elisa Ventura

    Full Text Available BACKGROUND: Fibronectin (FN is a multi-domain molecule involved in many cellular processes, including tissue repair, embryogenesis, blood clotting, and cell migration/adhesion. The biological activities of FN are mediated by exposed loops located mainly at the interdomain interfaces that interact with various molecules such as, but not only, integrins. Different FN isoforms arise from the alternative splicing of the pre-mRNA. In malignancies, the splicing pattern of FN pre-mRNA is altered; in particular, the FN isoform containing the extra-domain B (ED-B, a complete FN type III repeat constituted by 91 residues, is undetectable in normal adult tissues, but exhibits a much greater expression in fetal and tumor tissues, and is accumulate