WorldWideScience

Sample records for alu-derived intronic splicing

  1. 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.

  2. Inverse splicing of a group II intron.

    OpenAIRE

    Jarrell, K A

    1993-01-01

    I describe the self-splicing of an RNA that consists of exon sequences flanked by group II intron sequences. I find that this RNA undergoes accurate splicing in vitro, yielding an excised exon circle. This splicing reaction involves the joining of the 5' splice site at the end of an exon to the 3' splice site at the beginning of the same exon; thus, I term it inverse splicing. Inverse splicing provides a potential mechanism for exon scrambling, for exon deletion in alternative splicing pathwa...

  3. Group II Intron Self-Splicing.

    Science.gov (United States)

    Pyle, Anna Marie

    2016-07-01

    Group II introns are large, autocatalytic ribozymes that catalyze RNA splicing and retrotransposition. Splicing by group II introns plays a major role in the metabolism of plants, fungi, and yeast and contributes to genetic variation in many bacteria. Group II introns have played a major role in genome evolution, as they are likely progenitors of spliceosomal introns, retroelements, and other machinery that controls genetic variation and stability. The structure and catalytic mechanism of group II introns have recently been elucidated through a combination of genetics, chemical biology, solution biochemistry, and crystallography. These studies reveal a dynamic machine that cycles progressively through multiple conformations as it stimulates the various stages of splicing. A central active site, containing a reactive metal ion cluster, catalyzes both steps of self-splicing. These studies provide insights into RNA structure, folding, and catalysis, as they raise new questions about the behavior of RNA machines. PMID:27391926

  4. Kluyveromyces lactis maintains Saccharomyces cerevisiae intron-encoded splicing signals.

    OpenAIRE

    Deshler, J O; Larson, G P; Rossi, J J

    1989-01-01

    The actin (ACT) gene from the budding yeast Kluyveromyces lactis was cloned, and the nucleotide sequence was determined. The gene had a single intron 778 nucleotides in length which possessed the highly conserved splicing signals found in Saccharomyces cerevisiae introns. We demonstrated splicing of heterologous ACT transcripts in both K. lactis and S. cerevisiae.

  5. Ancient nature of alternative splicing and functions of introns

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Kemin; Salamov, Asaf; Kuo, Alan; Aerts, Andrea; Grigoriev, Igor

    2011-03-21

    Using four genomes: Chamydomonas reinhardtii, Agaricus bisporus, Aspergillus carbonarius, and Sporotricum thermophile with EST coverage of 2.9x, 8.9x, 29.5x, and 46.3x respectively, we identified 11 alternative splicing (AS) types that were dominated by intron retention (RI; biased toward short introns) and found 15, 35, 52, and 63percent AS of multiexon genes respectively. Genes with AS were more ancient, and number of AS correlated with number of exons, expression level, and maximum intron length of the gene. Introns with tendency to be retained had either stop codons or length of 3n+1 or 3n+2 presumably triggering nonsense-mediated mRNA decay (NMD), but introns retained in major isoforms (0.2-6percent of all introns) were biased toward 3n length and stop codon free. Stopless introns were biased toward phase 0, but 3n introns favored phase 1 that introduced more flexible and hydrophilic amino acids on both ends of introns which would be less disruptive to protein structure. We proposed a model in which minor RI intron could evolve into major RI that could facilitate intron loss through exonization.

  6. Functional characterisation of an intron retaining K+ transporter of barley reveals intron-mediated alternate splicing

    KAUST Repository

    Shahzad, K.

    2015-01-01

    Intron retention in transcripts and the presence of 5 and 3 splice sites within these introns mediate alternate splicing, which is widely observed in animals and plants. Here, functional characterisation of the K+ transporter, HvHKT2;1, with stably retained introns from barley (Hordeum vulgare) in yeast (Saccharomyces cerevisiae), and transcript profiling in yeast and transgenic tobacco (Nicotiana tabacum) is presented. Expression of intron-retaining HvHKT2;1 cDNA (HvHKT2;1-i) in trk1, trk2 yeast strain defective in K+ uptake restored growth in medium containing hygromycin in the presence of different concentrations of K+ and mediated hypersensitivity to Na+. HvHKT2;1-i produces multiple transcripts via alternate splicing of two regular introns and three exons in different compositions. HKT isoforms with retained introns and exon skipping variants were detected in relative expression analysis of (i) HvHKT2;1-i in barley under native conditions, (ii) in transgenic tobacco plants constitutively expressing HvHKT2;1-i, and (iii) in trk1, trk2 yeast expressing HvHKT2;1-i under control of an inducible promoter. Mixed proportions of three HKT transcripts: HvHKT2;1-e (first exon region), HvHKT2;1-i1 (first intron) and HvHKT2;1-i2 (second intron) were observed. The variation in transcript accumulation in response to changing K+ and Na+ concentrations was observed in both heterologous and plant systems. These findings suggest a link between intron-retaining transcripts and different splice variants to ion homeostasis, and their possible role in salt stress.

  7. Influence of intron length on interaction characters between post-spliced intron and its CDS in ribosomal protein genes

    Science.gov (United States)

    Zhao, Xiaoqing; Li, Hong; Bao, Tonglaga; Ying, Zhiqiang

    2012-09-01

    Many experiment evidences showed that sequence structures of introns and intron loss/gain can influence gene expression, but current mechanisms did not refer to the functions of post-spliced introns directly. We propose that postspliced introns play their functions in gene expression by interacting with their mRNA sequences and the interaction is characterized by the matched segments between introns and their CDS. In this study, we investigated the interaction characters with length series by improved Smith-Waterman local alignment software for the ribosomal protein genes in C. elegans and D. melanogaster. Our results showed that RF values of five intron groups are significantly high in the central non-conserved region and very low in 5'-end and 3'-end splicing region. It is interesting that the number of the optimal matched regions gradually increases with intron length. Distributions of the optimal matched regions are different for five intron groups. Our study revealed that there are more interaction regions between longer introns and their CDS than shorter, and it provides a positive pattern for regulating the gene expression.

  8. Alternative splicing mechanisms orchestrating post-transcriptional gene expression: intron retention and the intron-rich genome of apicomplexan parasites.

    Science.gov (United States)

    Lunghi, Matteo; Spano, Furio; Magini, Alessandro; Emiliani, Carla; Carruthers, Vern B; Di Cristina, Manlio

    2016-02-01

    Apicomplexan parasites including Toxoplasma gondii and Plasmodium species have complex life cycles that include multiple hosts and differentiation through several morphologically distinct stages requiring marked changes in gene expression. This review highlights emerging evidence implicating regulation of mRNA splicing as a mechanism to prime these parasites for rapid gene expression upon differentiation. We summarize the most important insights in alternative splicing including its role in regulating gene expression by decreasing mRNA abundance via 'Regulated Unproductive Splicing and Translation'. As a related but less well-understood mechanism, we discuss also our recent work suggesting a role for intron retention for precluding translation of stage specific isoforms of T. gondii glycolytic enzymes. We additionally provide new evidence that intron retention might be a widespread mechanism during parasite differentiation. Supporting this notion, recent genome-wide analysis of Toxoplasma and Plasmodium suggests intron retention is more pervasive than heretofore thought. These findings parallel recent emergence of intron retention being more prevalent in mammals than previously believed, thereby adding to the established roles in plants, fungi and unicellular eukaryotes. Deeper mechanistic studies of intron retention will provide important insight into its role in regulating gene expression in apicomplexan parasites and more general in eukaryotic organisms. PMID:26194054

  9. 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. PMID:24214997

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

    OpenAIRE

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

    1996-01-01

    The increased use of pure starter cultures in the wine industry has made it necessary to develop a rapid and simple identification system for yeast strains. A method based upon the PCR using oligonucleotide primers that are complementary to intron splice sites has been developed. Since most introns are not essential for gene function, introns have evolved with minimal constraint. By targeting these highly variable sequences, the PCR has proved to be very effective in uncovering polymorphisms ...

  11. 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. PMID:24751650

  12. 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...

  13. Class I self-splicing introns are found in the T-even bacteriophage family

    International Nuclear Information System (INIS)

    The thymidylate synthase gene (td) and ribonucleotide reductase B2 subunit gene (nrdB) EMBO both of bacteriophage T4 in origin, are procaryotic intron-containing protein-encoding genes. To screen for other procaryotic introns, southern hybridization analysis of several procaryotic genomes was carried out, using T4 phage td DNA restriction fragments and synthetic oligodeoxynucleotides defining strategic td exon and intron regions. Furthermore, the labeling pattern of total RNA with [α-32P]GTP, a typical reaction of self-splicing RNAs (class I), was examined. Experimental data implicate multiple self-splicing introns only in the T-even phages: five (1, 0.9, 0.83, 0.75 and 0.6 kb) in T4 and three (1, 0.9 and 0.75 kb) each in T2 and T6 phages. Northern hybridization analysis of total RNA extracted from T-even phage-infected cells confirms that the 1 kb RNA from each phage is in fact the excised intron segment from the precursor RNA transcribed from an intron-containing td gene in each case. This RNA cyclizes to form a contiguous circular molecule. The 0.6 kb RNA is most likely the T4 phage nrdB intron which seems to be absent from the corresponding gene in T2 and T6. The remaining RNA species are candidates for other self-splicing introns in these phages

  14. Differential GC Content between Exons and Introns Establishes Distinct Strategies of Splice-Site Recognition

    Directory of Open Access Journals (Sweden)

    Maayan Amit

    2012-05-01

    Full Text Available During evolution segments of homeothermic genomes underwent a GC content increase. Our analyses reveal that two exon-intron architectures have evolved from an ancestral state of low GC content exons flanked by short introns with a lower GC content. One group underwent a GC content elevation that abolished the differential exon-intron GC content, with introns remaining short. The other group retained the overall low GC content as well as the differential exon-intron GC content, and is associated with longer introns. We show that differential exon-intron GC content regulates exon inclusion level in this group, in which disease-associated mutations often lead to exon skipping. This group's exons also display higher nucleosome occupancy compared to flanking introns and exons of the other group, thus “marking” them for spliceosomal recognition. Collectively, our results reveal that differential exon-intron GC content is a previously unidentified determinant of exon selection and argue that the two GC content architectures reflect the two mechanisms by which splicing signals are recognized: exon definition and intron definition.

  15. Better late than early: delayed translation of intron-encoded endonuclease I-TevI is required for efficient splicing of its host group I intron.

    Science.gov (United States)

    Gibb, Ewan A; Edgell, David R

    2010-10-01

    The td group I intron interrupting the thymidylate synthase (TS) gene of phage T4 is a mobile intron that encodes the homing endonuclease I-TevI. Efficient RNA splicing of the intron is required to restore function of the TS gene, while expression of I-TevI from within the intron is required to initiate intron mobility. Three distinct layers of regulation temporally limit I-TevI expression to late in the T4 infective cycle, yet the biological rationale for stringent regulation has not been tested. Here, we deleted key control elements to deregulate I-TevI expression at early and middle times post T4 infection. Strikingly, we found that deregulation of I-TevI, or of a catalytically inactive variant, generated a thymidine-dependent phenotype that is caused by a reduction in td intron splicing. Prematurely terminating I-TevI translation restores td splicing, full-length TS synthesis, and rescues the thymidine-dependent phenotype. We suggest that stringent translational control of I-TevI evolved to prevent the ribosome from disrupting key structural elements of the td intron that are required for splicing and TS function at early and middle times post T4 infection. Analogous translational regulatory mechanisms in unrelated intron-open reading frame arrangements may also function to limit deleterious consequences on splicing and host gene function. PMID:20497330

  16. Learning to live together: mutualism between self-splicing introns and their hosts

    OpenAIRE

    Chalamcharla Venkata R; Edgell David R; Belfort Marlene

    2011-01-01

    Abstract Group I and II introns can be considered as molecular parasites that interrupt protein-coding and structural RNA genes in all domains of life. They function as self-splicing ribozymes and thereby limit the phenotypic costs associated with disruption of a host gene while they act as mobile DNA elements to promote their spread within and between genomes. Once considered purely selfish DNA elements, they now seem, in the light of recent work on the molecular mechanisms regulating bacter...

  17. Learning to live together: mutualism between self-splicing introns and their hosts

    Directory of Open Access Journals (Sweden)

    Chalamcharla Venkata R

    2011-04-01

    Full Text Available Abstract Group I and II introns can be considered as molecular parasites that interrupt protein-coding and structural RNA genes in all domains of life. They function as self-splicing ribozymes and thereby limit the phenotypic costs associated with disruption of a host gene while they act as mobile DNA elements to promote their spread within and between genomes. Once considered purely selfish DNA elements, they now seem, in the light of recent work on the molecular mechanisms regulating bacterial and phage group I and II intron dynamics, to show evidence of co-evolution with their hosts. These previously underappreciated relationships serve the co-evolving entities particularly well in times of environmental stress.

  18. Activating the branch-forming splicing pathway by reengineering the ribozyme component of a natural group II intron.

    Science.gov (United States)

    Monachello, Dario; Michel, François; Costa, Maria

    2016-03-01

    When assayed in vitro, group IIC self-splicing introns, which target bacterial Rho-independent transcription terminators, generally fail to yield branched products during splicing despite their possessing a seemingly normal branchpoint. Starting with intron O.i.I1 from Oceanobacillus iheyensis, whose crystallographically determined structure lacks branchpoint-containing domain VI, we attempted to determine what makes this intron unfit for in vitro branch formation. A major factor was found to be the length of the helix at the base of domain VI: 4 base pairs (bp) are required for efficient branching, even though a majority of group IIC introns have a 3-bp helix. Equally important for lariat formation is the removal of interactions between ribozyme domains II and VI, which are specific to the second step of splicing. Conversely, mismatching of domain VI and its proposed first-step receptor in subdomain IC1 was found to be detrimental; these data suggest that the intron-encoded protein may promote branch formation partly by modulating the equilibrium between conformations specific to the first and second steps of splicing. As a practical application, we show that by making just two changes to the O.i.I1 ribozyme, it is possible to generate sufficient amounts of lariat intron for the latter to be purified and used in kinetic assays in which folding and reaction are uncoupled. PMID:26769855

  19. Group II intron splicing in Escherichia coli: phenotypes of cis-acting mutations resemble splicing defects observed in organelle RNA processing.

    OpenAIRE

    Holländer, V; Kück, U

    1999-01-01

    The mitochondrial group IIB intron rI1, from the green algae Scenedesmus obliquus ' LSUrRNA gene, has been introduced into the lacZ gene encoding beta-galacto-sidase. After DNA-mediated transformation of the recombinant lacZ gene into Escherichia coli, we observed correct splicing of the chimeric precursor RNA in vivo. In contrast to autocatalytic in vitro self-splicing, intron processing in vivo is independent of the growth temperature, suggesting that in E.coli, trans -acting factors are in...

  20. PMD patient mutations reveal a long-distance intronic interaction that regulates PLP1/DM20 alternative splicing

    OpenAIRE

    Taube, Jennifer R.; Sperle, Karen; Banser, Linda; Seeman, Pavel; Cavan, Barbra Charina V.; Garbern, James Y.; Hobson, Grace M.

    2014-01-01

    Alternative splicing of the proteolipid protein 1 gene (PLP1) produces two forms, PLP1 and DM20, due to alternative use of 5′ splice sites with the same acceptor site in intron 3. The PLP1 form predominates in central nervous system RNA. Mutations that reduce the ratio of PLP1 to DM20, whether mutant or normal protein is formed, result in the X-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD). We investigated the ability of sequences throughout PLP1 intron 3 to regulate alternative sp...

  1. Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions

    International Nuclear Information System (INIS)

    Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human cancer gene in JAR cells. We screened 10 different 15 mer PNAs targeting intron2 at both the 5' - and the 3'-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512) targeting the 3'-splice site of intron3 with a complementarity of 4 bases to intron3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT). We show that several of these PNAs effectively inhibit the splicing thereby producing a larger mRNA still containing intron2, while skipping of exon3 was not observed by any of these PNAs. The most effective PNA (PNA2406) targeting the 3'-splice site of intron2 had a complementarity of 4 bases to intron2 and 11 bases to exon3. PNA (2512) targeting the 3'-splice site of intron3 induced both splicing inhibition (intron3 skipping) and skipping of exon4. Furthermore, treatment of JAR cells with this PNA resulted in a reduction in the level of MDM2 protein and a concomitant increase in the level of tumor suppressor p53. In addition, a combination of this PNA with CPT inhibited cell growth more than CPT alone. We have identified several PNAs targeting the 5'- or 3'-splice sites in intron2 or the 3'-splice site of intron3 of mdm2 pre-mRNA which can inhibit splicing. Antisense targeting of splice junctions of mdm2 pre-mRNA may be a powerful method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs

  2. A One-Step PCR Method for Detecting the First Base of Splice Donor of Wx Intron 1 in Rice

    Institute of Scientific and Technical Information of China (English)

    MAO Xing-xue; LIU Yan-zhuo; XIAO Xin; CHEN Jian-wei; LUO Wen-yong; LI Xiao-fang

    2004-01-01

    A new method of one-step PGR was devised for detecting the first nucleotide in the splice donor site of Wx intron 1.compared to the regular PCR-Acc I method, the method can produce the same result for detecting +1 nucleotide of Wx intron 1.The reliability of the new method was confirmed with 30 rice varieties. The new technique is more convenient and cheaper than the regular PCR-Acc I method, and could be widely deploded in rice molecular marker assistant selection.

  3. Using intron splicing trick for preferential gene expression in transduced cells: an approach for suicide gene therapy.

    Science.gov (United States)

    Pourzadegan, F; Shariati, L; Taghizadeh, R; Khanahmad, H; Mohammadi, Z; Tabatabaiefar, M A

    2016-01-01

    Suicide gene therapy is one of the most innovative approaches in which a potential toxic gene is delivered to the targeted cancer cell by different target delivery methods. We constructed a transfer vector to express green fluorescent protein (GFP) in transduced cells but not in packaging cells. We placed gfp under the control of the cytomegalovirus (CMV) promoter, which is positioned between the two long-terminal repeats in reverse direction. The intron-2 sequence of the human beta globin gene with two poly-A signals and several stop codons on the antisense strand was placed on the leading strand between the CMV promoter and gfp. For lentiviral production, the HEK293T and line were co-transfected with the PMD2G, psPAX2 and pLentiGFP-Ins2 plasmids. The HEK293T and line were transduced with this virus. PCR was performed for evaluation of intron splicing in transduced cells. The GFP expression was seen in 65% of the cells transduced. The PCR amplification of the genomic DNA of transduced cells confirmed the splicing of intron 2. The strategy is significant to accomplish our goal for preserving the packaging cells from the toxic gene expression during viral assembly and the resultant reduction in viral titration. Also it serves to address several other issues in the gene therapy. PMID:26679755

  4. Inclusion of the Central Exon of Parvovirus B19 Precursor mRNA Is Determined by Multiple Splicing Enhancers in both the Exon and the Downstream Intron

    OpenAIRE

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

    2010-01-01

    Alternative splicing of the precursor mRNA (pre-mRNA) of human parvovirus B19 (B19V) plays a key role in posttranscriptional regulation of B19V gene expression. We report that the central exon of the B19V pre-mRNA is defined by three GAA motif-containing exonic splicing enhancers and a G/GU-rich intronic splicing enhancer that lies adjacent to the second donor site. Moreover, targeting of morpholino antisense oligonucleotides to the two splicing enhancers surrounding the second donor site led...

  5. Abiotic stresses affect differently the intron splicing and expression of chloroplast genes in coffee plants (Coffea arabica) and rice (Oryza sativa).

    Science.gov (United States)

    Nguyen Dinh, Sy; Sai, Than Zaw Tun; Nawaz, Ghazala; Lee, Kwanuk; Kang, Hunseung

    2016-08-20

    Despite the increasing understanding of the regulation of chloroplast gene expression in plants, the importance of intron splicing and processing of chloroplast RNA transcripts under stress conditions is largely unknown. Here, to understand how abiotic stresses affect the intron splicing and expression patterns of chloroplast genes in dicots and monocots, we carried out a comprehensive analysis of the intron splicing and expression patterns of chloroplast genes in the coffee plant (Coffea arabica) as a dicot and rice (Oryza sativa) as a monocot under abiotic stresses, including drought, cold, or combined drought and heat stresses. The photosynthetic activity of both coffee plants and rice seedlings was significantly reduced under all stress conditions tested. Analysis of the transcript levels of chloroplast genes revealed that the splicing of tRNAs and mRNAs in coffee plants and rice seedlings were significantly affected by abiotic stresses. Notably, abiotic stresses affected differently the splicing of chloroplast tRNAs and mRNAs in coffee plants and rice seedlings. The transcript levels of most chloroplast genes were markedly downregulated in both coffee plants and rice seedlings upon stress treatment. Taken together, these results suggest that coffee and rice plants respond to abiotic stresses via regulating the intron splicing and expression of different sets of chloroplast genes. PMID:27448724

  6. Insertion of a self-splicing intron into the mtDNA of atriploblastic animal

    Energy Technology Data Exchange (ETDEWEB)

    Valles, Y.; Halanych, K.; Boore, J.L.

    2006-04-14

    Nephtys longosetosa is a carnivorous polychaete worm that lives in the intertidal and subtidal zones with worldwide distribution (pleijel&rouse2001). Its mitochondrial genome has the characteristics typical of most metazoans: 37 genes; circular molecule; almost no intergenic sequence; and no significant gene rearrangements when compared to other annelid mtDNAs (booremoritz19981995). Ubiquitous features as small intergenic regions and lack of introns suggested that metazoan mtDNAs are under strong selective pressures to reduce their genome size allowing for faster replication requirements (booremoritz19981995Lynch2005). Yet, in 1996 two type I introns were found in the mtDNA of the basal metazoan Metridium senile (FigureX). Breaking a long-standing rule (absence of introns in metazoan mtDNA), this finding was later supported by the further presence of group I introns in other cnidarians. Interestingly, only the class Anthozoa within cnidarians seems to harbor such introns. Although several hundreds of triploblastic metazoan mtDNAs have been sequenced, this study is the first evidence of mitochondrial introns in triploblastic metazoans. The cox1 gene of N. longosetosa has an intron of almost 2 kbs in length. This finding represents as well the first instance of a group II intron (anthozoans harbor group I introns) in all metazoan lineages. Opposite trends are observed within plants, fungi and protist mtDNAs, where introns (both group I and II) and other non-coding sequences are widespread. Plant, fungal and protist mtDNA structure and organization differ enormously from that of metazoan mtDNA. Both, plant and fungal mtDNA are dynamic molecules that undergo high rates of recombination, contain long intergenic spacer regions and harbor both group I and group II introns. However, as metazoans they have a conserved gene content. Protists, on the other hand have a striking variation of gene content and introns that account for the genome size variation. In contrast to

  7. Compound heterozygous mutations in the noncoding RNU4ATAC cause Roifman Syndrome by disrupting minor intron splicing

    Science.gov (United States)

    Merico, Daniele; Roifman, Maian; Braunschweig, Ulrich; Yuen, Ryan K. C.; Alexandrova, Roumiana; Bates, Andrea; Reid, Brenda; Nalpathamkalam, Thomas; Wang, Zhuozhi; Thiruvahindrapuram, Bhooma; Gray, Paul; Kakakios, Alyson; Peake, Jane; Hogarth, Stephanie; Manson, David; Buncic, Raymond; Pereira, Sergio L.; Herbrick, Jo-Anne; Blencowe, Benjamin J.; Roifman, Chaim M.; Scherer, Stephen W.

    2015-01-01

    Roifman Syndrome is a rare congenital disorder characterized by growth retardation, cognitive delay, spondyloepiphyseal dysplasia and antibody deficiency. Here we utilize whole-genome sequencing of Roifman Syndrome patients to reveal compound heterozygous rare variants that disrupt highly conserved positions of the RNU4ATAC small nuclear RNA gene, a minor spliceosome component that is essential for minor intron splicing. Targeted sequencing confirms allele segregation in six cases from four unrelated families. RNU4ATAC rare variants have been recently reported to cause microcephalic osteodysplastic primordial dwarfism, type I (MOPD1), whose phenotype is distinct from Roifman Syndrome. Strikingly, all six of the Roifman Syndrome cases have one variant that overlaps MOPD1-implicated structural elements, while the other variant overlaps a highly conserved structural element not previously implicated in disease. RNA-seq analysis confirms extensive and specific defects of minor intron splicing. Available allele frequency data suggest that recessive genetic disorders caused by RNU4ATAC rare variants may be more prevalent than previously reported. PMID:26522830

  8. Slow formation of a pseudoknot structure is rate limiting in the productive co-transcriptional folding of the self-splicing Candida intron.

    Science.gov (United States)

    Zhang, Libin; Bao, Penghui; Leibowitz, Michael J; Zhang, Yi

    2009-11-01

    Pseudoknots play critical roles in packing the active structure of various functional RNAs. The importance of the P3-P7 pseudoknot in refolding of group I intron ribozymes has been recently appreciated, while little is known about the pseudoknot function in co-transcriptional folding. Here we used the Candida group I intron as a model to address the question. We show that co-transcriptional folding of the active self-splicing intron is twice as fast as refolding. The P3-P7 pseudoknot folds slowly during co-transcriptional folding at a rate constant similar to the folding of the active ribozyme, and folding of both P3-P7 and P1-P10 pseudoknots are inhibited by antisense oligonucleotides. We conclude that when RNA folding is coupled with transcription, formation of pseudoknot structures dominates the productive folding pathway and serves as a rate-limiting step in producing the self-splicing competent Candida intron. PMID:19710184

  9. Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis.

    OpenAIRE

    Boudvillain, M; Pyle, A M

    1998-01-01

    Group II introns are self-splicing RNA molecules that are of considerable interest as ribozymes, mobile genetic elements and examples of folded RNA. Although these introns are among the most common ribozymes, little is known about the chemical and structural determinants for their reactivity. By using nucleotide analog interference mapping (NAIM), it has been possible to identify the nucleotide functional groups (Rp phosphoryls, 2'-hydroxyls, guanosine exocyclic amines, adenosine N7 and N6) t...

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

    OpenAIRE

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

    1993-01-01

    In a previous study we found that a Tay-Sachs disease (TSD) causing mutation in the intron 9 donor splice site of the HEXA gene occurs at high frequency in non-Jewish patients and carriers from the British Isles. It was found more frequently in subjects of Irish, Scottish, and Welsh origin compared with English origin (63% and 31% respectively). We have now tested, in a blind study, 26 American TSD carriers and 28 non-carriers who have British ancestry for the intron 9 splice site mutation. S...

  11. EMPTY PERICARP16 is required for mitochondrial nad2 intron 4 cis-splicing, complex I assembly and seed development in maize.

    Science.gov (United States)

    Xiu, Zhihui; Sun, Feng; Shen, Yun; Zhang, Xiaoyan; Jiang, Ruicheng; Bonnard, Géraldine; Zhang, Jianhua; Tan, Bao-Cai

    2016-02-01

    In higher plants, chloroplast and mitochondrial transcripts contain a number of group II introns that need to be precisely spliced before translation into functional proteins. However, the mechanism of splicing and the factors involved in this process are not well understood. By analysing a seed mutant in maize, we report here the identification of Empty pericarp16 (Emp16) that is required for splicing of nad2 intron 4 in mitochondria. Disruption of Emp16 function causes developmental arrest in the embryo and endosperm, giving rise to an empty pericarp phenotype in maize. Differentiation of the basal endosperm transfer layer cells is severely affected. Molecular cloning indicates that Emp16 encodes a P-type pentatricopeptide repeat (PPR) protein with 11 PPR motifs and is localized in the mitochondrion. Transcript analysis revealed that mitochondrial nad2 intron 4 splicing is abolished in the emp16 mutants, leading to severely reduced assembly and activity of complex I. In response, the mutant dramatically increases the accumulation of mitochondrial complex III and the expression of alternative oxidase AOX2. These results imply that EMP16 is specifically required for mitochondrial nad2 intron 4 cis-splicing and is essential for complex I assembly and embryogenesis and development endosperm in maize. PMID:26764126

  12. Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Eysturskard, Jonhard; Nielsen, Peter E

    2010-01-01

    ABSTRACT: BACKGROUND: Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human...... cancer gene in JAR cells. METHODS: We screened 10 different 15 mer PNAs targeting intron2 at both the 5;- and the 3;-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512) targeting the 3;-splice site of intron3 with a complementarity of 4 bases to intron......3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT). RESULTS: We show that several of these PNAs effectively...

  13. Slow formation of a pseudoknot structure is rate limiting in the productive co-transcriptional folding of the self-splicing Candida intron

    OpenAIRE

    Zhang, Libin; Bao, Penghui; Leibowitz, Michael J.; Zhang, Yi

    2009-01-01

    Pseudoknots play critical roles in packing the active structure of various functional RNAs. The importance of the P3–P7 pseudoknot in refolding of group I intron ribozymes has been recently appreciated, while little is known about the pseudoknot function in co-transcriptional folding. Here we used the Candida group I intron as a model to address the question. We show that co-transcriptional folding of the active self-splicing intron is twice as fast as refolding. The P3–P7 pseudoknot folds sl...

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

    Directory of Open Access Journals (Sweden)

    Jitra Kriangkum

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

  15. 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...... synaptic weights of the neural networks trained to identify UTR donor sites. Conventional splice site prediction methods perform poorly in UTRs because the reading frame pattern is absent. The NetUTR method presented here performs 2-.3-fold better compared with NetGene2 and GenScan in 5' UTRs. We also...... tested the 5' UTR trained method on protein coding regions, and discovered, surprisingly, that it works quite well (although it cannot compete with NetGene2). This indicates that the local splicing pattern in UTRs and coding regions is largely the same. The NetUTR method is made publicly available at www.cbs.dtu.dk/services/NetUTR....

  16. Effects of intronic mutations in the LDLR gene on pre-mRNA splicing: Comparison of wet-lab and bioinformatics analyses

    NARCIS (Netherlands)

    O.L. Holla; S. Nakken; M. Mattingsdal; T. Ranheim; K.E. Berge; J.C. Defesche; T.P. Leren

    2009-01-01

    Screening for mutations in the low density lipoprotein receptor (LDLR) gene has identified more than 1000 mutations as the cause of familial hypercholesterolemia (FH). In addition, numerous intronic mutations with uncertain effects on pre-mRNA splicing have also been identified. In this study, we ha

  17. Archaeal rRNA operons, intron splicing and homing endonucleases, RNA polymerase operons and phylogeny

    DEFF Research Database (Denmark)

    Garrett, Roger Antony; Aagaard, Claus Sindbjerg; Andersen, Morten; Dalgaard, Jacob; Lykke-Andersen, Jens; Phan, Hoa T.N.; Trevisanato, Siro; Østergaard, Laust; Larsen, Niels; Leffers, Henrik

    1994-01-01

    Over the past decade our laboratory has had a strong interest in defining the phylogenetic status of the archaea. This has involved determining and analysing the sequences of operons of both rRNAs and RNA polymerases and it led to the discovery of the first archaeal rRNA intron. What follows is a...

  18. The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover.

    Directory of Open Access Journals (Sweden)

    Huei-Mei Chen

    Full Text Available The polyA tails of mRNAs are monitored by the exosome as a quality control mechanism. We find that fission yeast, Schizosaccharomyces pombe, adopts this RNA quality control mechanism to regulate a group of 30 or more meiotic genes at the level of both splicing and RNA turnover. In vegetative cells the RNA binding protein Mmi1 binds to the primary transcripts of these genes. We find the novel motif U(U/C/GAAAC highly over-represented in targets of Mmi1. Mmi1 can specifically regulate the splicing of particular introns in a transcript: it inhibits the splicing of introns that are in the vicinity of putative Mmi1 binding sites, while allowing the splicing of other introns that are far from such sites. In addition, binding of Mmi1, particularly near the 3' end, alters 3' processing to promote extremely long polyA tails of up to a kilobase. The hyperadenylated transcripts are then targeted for degradation by the nuclear exonuclease Rrp6. The nuclear polyA binding protein Pab2 assists this hyperadenylation-mediated RNA decay. Rrp6 also targets other hyperadenylated transcripts, which become hyperadenylated in an unknown, but Mmi1-independent way. Thus, hyperadenylation may be a general signal for RNA degradation. In addition, binding of Mmi1 can affect the efficiency of 3' cleavage. Inactivation of Mmi1 in meiosis allows meiotic expression, through splicing and RNA stabilization, of at least 29 target genes, which are apparently constitutively transcribed.

  19. Splicing of Yeast aI5β Group I Intron Requires SUV3 to Recycle MRS1 via Mitochondrial Degradosome-promoted Decay of Excised Intron Ribonucleoprotein (RNP)*

    OpenAIRE

    Turk, Edward M.; Caprara, Mark G.

    2010-01-01

    Yeast Suv3p is a member of the DEXH/D box family of RNA helicases and is a critical component of the mitochondrial degradosome, which also includes a 3′ → 5′ exonuclease, Dss1p. Defects in the degradosome result in accumulation of aberrant transcripts, unprocessed transcripts, and excised group I introns. In addition, defects in SUV3 result in decreased splicing of the aI5β and bI3 group I introns. Whereas a role for Suv3p in RNA degradation is well established, the function of Suv3p in splic...

  20. Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

    Energy Technology Data Exchange (ETDEWEB)

    Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo; Lersch,Robert A.; Gee, Sherry L.; Hou, Victor C.; Lo, Annie J.; Short, Sarah A.; Chasis, Joel A.; Winkelmann, John C.; Conboy, John G.

    2006-03-01

    Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.

  1. Modeling study on the cleavage step of the self-splicing reaction in group I introns

    Science.gov (United States)

    Setlik, R. F.; Garduno-Juarez, R.; Manchester, J. I.; Shibata, M.; Ornstein, R. L.; Rein, R.

    1993-01-01

    A three-dimensional model of the Tetrahymena thermophila group I intron is used to further explore the catalytic mechanism of the transphosphorylation reaction of the cleavage step. Based on the coordinates of the catalytic core model proposed by Michel and Westhof (Michel, F., Westhof, E. J. Mol. Biol. 216, 585-610 (1990)), we first converted their ligation step model into a model of the cleavage step by the substitution of several bases and the removal of helix P9. Next, an attempt to place a trigonal bipyramidal transition state model in the active site revealed that this modified model for the cleavage step could not accommodate the transition state due to insufficient space. A lowering of P1 helix relative to surrounding helices provided the additional space required. Simultaneously, it provided a better starting geometry to model the molecular contacts proposed by Pyle et al. (Pyle, A. M., Murphy, F. L., Cech, T. R. Nature 358, 123-128. (1992)), based on mutational studies involving the J8/7 segment. Two hydrated Mg2+ complexes were placed in the active site of the ribozyme model, using the crystal structure of the functionally similar Klenow fragment (Beese, L.S., Steitz, T.A. EMBO J. 10, 25-33 (1991)) as a guide. The presence of two metal ions in the active site of the intron differs from previous models, which incorporate one metal ion in the catalytic site to fulfill the postulated roles of Mg2+ in catalysis. The reaction profile is simulated based on a trigonal bipyramidal transition state, and the role of the hydrated Mg2+ complexes in catalysis is further explored using molecular orbital calculations.

  2. Interrupted thymidylate synthase gene of bacteriophages T2 and T6 and other potential self-splicing introns in the T-even bacteriophages

    International Nuclear Information System (INIS)

    Southern hybridization analyses of procaryotic DNA from Escherchia coli, λ bacteriophage, and T1 to T7 phages were carried out. The hybridization probes used consisted of DNA restriction fragments derived from the T4 phage intron-containing thymidylate synthase gene (td) and short synthetic oligodeoxynucleotides defining specific exon and intron regions of the gene. It was shown that intact as well as restricted DNA from the T-even phages hybridized not only to both T4 phage td intron- and exon-specific probes but also to probes defining the td 5' (exon I-intron) and 3' (intron-exon II) presplice junctions. These data strongly suggest that, analogous to the T4 phage, only the T2 and T6 phages among the procaryotes tested contain interrupted td genes. The td intervening sequence in each phage is roughly 1 kilobase pair (kb) in size and interrupts the td gene at a site analogous to that in the T4 phage. This was confirmed by data from Northern (RNA) hybridization analysis of td-specific in vitro transcripts of these phage DNAs. [α-32P]GTP in vitro labeling of total RNA from T4 phage-infected cells produced five species of labeled RNAs that were 1, 0.9, 0.83, 0.75, and 0.6 kb in size. Only the 1-, 0.9-, and 0.75-kb species were labeled in RNA from T2- or T6-infected cells. The commonly present 1-kb RNA is the excised td intron, which exists in both linear and circular forms in the respective T-even-phage-infected cells, while the 0.6-kb RNA unique to T4 may be the excised intron derived from the ribonucleotide reductase small subunit gene (nrdB) of the phage. The remaining labeled RNA species are likely candidates for other self-splicing introns

  3. Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS Gene

    Directory of Open Access Journals (Sweden)

    Yoko Nakajima

    2016-01-01

    Full Text Available Dihydropyrimidinase (DHP deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

  4. Introns in higher plant genes

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The intron is an important component of eukaryotic gene. Extensive studies have been conducted to get a better understanding of its structure and function. This paper presents a brief review of the structure and function of introns in higher plant genes. It is shown that higher plant introns possess structural properties shared by all eukaryotic introns, however, they also exhibit a striking degree of diversity. The process of intron splicing in higher plant genes involves interaction between multiple cis-acting elements and trans-acting factors, such as 5′ splicing site, 3′ splicing site and many protein factors. The process of intron splicing is an important level at which gene expression is regulated. Especially alternative splicing of intron can regulate time and space of gene expression. In addition, some introns in higher plant genes also regulate gene expression by affecting the pattern of gene expression, enhancing the level of gene expression and driving the gene expression.

  5. Cross-kingdom patterns of alternative splicing and splice recognition

    OpenAIRE

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

    2007-01-01

    Background: Variations in transcript splicing can reveal how eukaryotes recognize intronic splice sites. Retained introns (RIs) commonly appear when the intron definition (ID) mechanism of splice site recognition inconsistently identifies intron-exon boundaries, and cassette exons (CEs) are often caused by variable recognition of splice junctions by the exon definition (ED) mechanism. We have performed a comprehensive survey of alternative splicing across 42 eukaryotes to gain ins...

  6. Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis.

    Science.gov (United States)

    Boudvillain, M; Pyle, A M

    1998-12-01

    Group II introns are self-splicing RNA molecules that are of considerable interest as ribozymes, mobile genetic elements and examples of folded RNA. Although these introns are among the most common ribozymes, little is known about the chemical and structural determinants for their reactivity. By using nucleotide analog interference mapping (NAIM), it has been possible to identify the nucleotide functional groups (Rp phosphoryls, 2'-hydroxyls, guanosine exocyclic amines, adenosine N7 and N6) that are most important for composing the catalytic core of the intron. The majority of interference effects occur in clusters located within the two catalytically essential Domains 1 and 5 (D1 and D5). Collectively, the NAIM results indicate that key tetraloop-receptor interactions display a specific chemical signature, that the epsilon-epsilon' interaction includes an elaborate array of additional features and that one of the most important core structures is an uncharacterized three-way junction in D1. By combining NAIM with site-directed mutagenesis, a new tertiary interaction, kappa-kappa', was identified between this region and the most catalytically important section of D5, adjacent to the AGC triad in stem 1. Together with the known zeta-zeta' interaction, kappa-kappa' anchors D5 firmly into the D1 scaffold, thereby presenting chemically essential D5 functionalities for participation in catalysis. PMID:9843513

  7. Diverse splicing patterns of exonized Alu elements in human tissues.

    Directory of Open Access Journals (Sweden)

    Lan Lin

    Full Text Available Exonization of Alu elements is a major mechanism for birth of new exons in primate genomes. Prior analyses of expressed sequence tags show that almost all Alu-derived exons are alternatively spliced, and the vast majority of these exons have low transcript inclusion levels. In this work, we provide genomic and experimental evidence for diverse splicing patterns of exonized Alu elements in human tissues. Using Exon array data of 330 Alu-derived exons in 11 human tissues and detailed RT-PCR analyses of 38 exons, we show that some Alu-derived exons are constitutively spliced in a broad range of human tissues, and some display strong tissue-specific switch in their transcript inclusion levels. Most of such exons are derived from ancient Alu elements in the genome. In SEPN1, mutations of which are linked to a form of congenital muscular dystrophy, the muscle-specific inclusion of an Alu-derived exon may be important for regulating SEPN1 activity in muscle. Realtime qPCR analysis of this SEPN1 exon in macaque and chimpanzee tissues indicates human-specific increase in its transcript inclusion level and muscle specificity after the divergence of humans and chimpanzees. Our results imply that some Alu exonization events may have acquired adaptive benefits during the evolution of primate transcriptomes.

  8. 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).

  9. 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.

  10. The unusual 23S rRNA gene of Coxiella burnetii: two self-splicing group I introns flank a 34-base-pair exon, and one element lacks the canonical omegaG.

    Science.gov (United States)

    Raghavan, Rahul; Miller, Scott R; Hicks, Linda D; Minnick, Michael F

    2007-09-01

    We describe the presence and characteristics of two self-splicing group I introns in the sole 23S rRNA gene of Coxiella burnetii. The two group I introns, Cbu.L1917 and Cbu.L1951, are inserted at sites 1917 and 1951 (Escherichia coli numbering), respectively, in the 23S rRNA gene of C. burnetii. Both introns were found to be self-splicing in vivo and in vitro even though the terminal nucleotide of Cbu.L1917 is adenine and not the canonical conserved guanine, termed OmegaG, found in Cbu.L1951 and all other group I introns described to date. Predicted secondary structures for both introns were constructed and revealed that Cbu.L1917 and Cbu.L1951 were group IB2 and group IA3 introns, respectively. We analyzed strains belonging to eight genomic groups of C. burnetii to determine sequence variation and the presence or absence of the elements and found both introns to be highly conserved (>/=99%) among them. Although phylogenetic analysis did not identify the specific identities of donors, it indicates that the introns were likely acquired independently; Cbu.L1917 was acquired from other bacteria like Thermotoga subterranea and Cbu.L1951 from lower eukaryotes like Acanthamoeba castellanii. We also confirmed the fragmented nature of mature 23S rRNA in C. burnetii due to the presence of an intervening sequence. The presence of three selfish elements in C. burnetii's 23S rRNA gene is very unusual for an obligate intracellular bacterium and suggests a recent shift to its current lifestyle from a previous niche with greater opportunities for lateral gene transfer. PMID:17644584

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

    OpenAIRE

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

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipp...

  12. 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.

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

    Science.gov (United States)

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

    1994-01-01

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

  14. 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.

  15. SRSF1 (SRp30a) regulates the alternative splicing of caspase 9 via a novel intronic splicing enhancer affecting the chemotherapeutic sensitivity of non-small cell lung cancer cells

    OpenAIRE

    Shultz, Jacqueline C.; Rachel W Goehe; Murudkar, Charuta S.; Wijesinghe, Dayanjan S.; Mayton, Eric K.; Massiello, Autumn; Hawkins, Amy J.; Mukerjee, Prabhat; Pinkerman, Ryan L.; Park, Margaret A; Chalfant, Charles E.

    2011-01-01

    Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology with the alternative pre-mRNA processing of caspase 9 as one example. In this study, we delve into the underlying molecular mechanisms that regulate the alternative splicing of caspase 9. Specifically, the pre-mRNA sequence of caspase 9 was analyzed for RNA cis-elements known to interact with SRSF1, a required enhancer for caspase 9 RNA splicing. This analysis revealed thirteen p...

  16. Reenacting the birth of an intron

    Energy Technology Data Exchange (ETDEWEB)

    Hellsten, Uffe; Aspden, Julie L.; Rio, Donald C.; Rokhsar, Daniel S.

    2011-07-01

    An intron is an extended genomic feature whose function requires multiple constrained positions - donor and acceptor splice sites, a branch point, a polypyrimidine tract and suitable splicing enhancers - that may be distributed over hundreds or thousands of nucleotides. New introns are therefore unlikely to emerge by incremental accumulation of functional sub-elements. Here we demonstrate that a functional intron can be created de novo in a single step by a segmental genomic duplication. This experiment recapitulates in vivo the birth of an intron that arose in the ancestral jawed vertebrate lineage nearly half a billion years ago.

  17. Group I introns - moving in new directions

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Johansen, Steinar

    2009-01-01

    Group I introns are genetic elements interrupting functional genes. They are removed from precursors at the RNA level and most catalyze their own splicing. The catalytic part of these constitutes one of the major classes of catalytic RNAs, the group I ribozymes. However, group I introns have a lot...... more to offer than their own elimination by splicing. Intron RNA can circularize in at least three different ways and introns are mobile both at the DNA and RNA level. Some group I introns have a very complex organization incorporating functional genes and other sequence elements and have established...... deep relationships with their host genomes. Finally, group I introns can develop into new ribozymes with new biological functions...

  18. Statistical characteristics of eukaryotic intron database

    Institute of Scientific and Technical Information of China (English)

    HE Miao; LI Jidong; ZHANG Shanghong

    2006-01-01

    A database called eukaryotic intron database (EID) was developed based on the data from GenBank.Studies on the statistical characteristics of EID show that there were 103,848 genes,478,484 introns,and 582,332 exons,with an average of 4.61 introns and 5.61 exons per gene.Introns of 40-120 nt in length were abundant in the database.Results of the statistical analysis on the data from nine model species showed that in eukaryotes,higher species do not necessarily have more introns or exons in a gene than lower species.Furthermore,characteristics of EID,such as intron phase,distribution of different splice sites,and the relationship between genome size and intron proportion or intron density,have been studied.

  19. The Brown Algae Pl.LSU/2 Group II Intron-Encoded Protein Has Functional Reverse Transcriptase and Maturase Activities

    OpenAIRE

    Madeleine Zerbato; Nathalie Holic; Sophie Moniot-Frin; Dina Ingrao; Anne Galy; Javier Perea

    2013-01-01

    Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is...

  20. The Trypanosoma brucei La protein is a candidate poly(U) shield that impacts spliced leader RNA maturation and tRNA intron removal

    Czech Academy of Sciences Publication Activity Database

    Trantírková, Silvie; Paris, Zdeněk; Sturm, N. R.; Campbell, D. A.; Lukeš, Julius

    2005-01-01

    Roč. 35, č. 4 (2005), s. 359-366. ISSN 0020-7519 R&D Projects: GA AV ČR IAA5022302 Grant ostatní: NIH(US) AI34536; NIH(US) AI056034 Institutional research plan: CEZ:AV0Z60220518 Keywords : splicing * Trypanosoma * RNA interference Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.346, year: 2005

  1. Bifunctional RNAs Targeting the Intronic Splicing Silencer N1 Increase SMN Levels and Reduce Disease Severity in an Animal Model of Spinal Muscular Atrophy

    OpenAIRE

    Osman, Erkan Y; Yen, Pei-Fen; Lorson, Christian L.

    2011-01-01

    Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of survival motor neuron-1 (SMN1). A nearly identical copy gene, SMN2, is present in all SMA patients. Although the SMN2 coding sequence has the potential to produce full-length SMN, nearly 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein. SMN2, however, is an excellent therapeutic target. Previously, we developed antisense-based oligonucleotides (bifunctional RNAs) that specifi...

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

    OpenAIRE

    Schwartz, Schraga; Silva, João(CFTP, Departamento de Física, Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais 1, 1049, Lisboa, Portugal); Burstein, David; Pupko, Tal; Eyras, Eduardo; Ast, Gil

    2008-01-01

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

  3. Conserved RNA secondary structures promote alternative splicing

    OpenAIRE

    Shepard, PJ; Hertel, KJ

    2008-01-01

    Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. Alternative splicing in higher eukaryotes results in the generation of multiple protein isoforms from gene transcripts. The extensive alternative splicing observed implies a flexibility of the spliceosome to identify exons within a given pre-mRNA. To reach this flexibility, splice-site selection in higher eukaryotes has evolved to depend on multiple parameters such as splice-site stren...

  4. The origin of introns and their role in eukaryogenesis: a compromise solution to the introns-early versus introns-late debate?

    Directory of Open Access Journals (Sweden)

    Koonin Eugene V

    2006-08-01

    Full Text Available Abstract Background Ever since the discovery of 'genes in pieces' and mRNA splicing in eukaryotes, origin and evolution of spliceosomal introns have been considered within the conceptual framework of the 'introns early' versus 'introns late' debate. The 'introns early' hypothesis, which is closely linked to the so-called exon theory of gene evolution, posits that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. Under this scenario, the absence of spliceosomal introns in prokaryotes is considered to be a result of "genome streamlining". The 'introns late' hypothesis counters that spliceosomal introns emerged only in eukaryotes, and moreover, have been inserted into protein-coding genes continuously throughout the evolution of eukaryotes. Beyond the formal dilemma, the more substantial side of this debate has to do with possible roles of introns in the evolution of eukaryotes. Results I argue that several lines of evidence now suggest a coherent solution to the introns-early versus introns-late debate, and the emerging picture of intron evolution integrates aspects of both views although, formally, there seems to be no support for the original version of introns-early. Firstly, there is growing evidence that spliceosomal introns evolved from group II self-splicing introns which are present, usually, in small numbers, in many bacteria, and probably, moved into the evolving eukaryotic genome from the α-proteobacterial progenitor of the mitochondria. Secondly, the concept of a primordial pool of 'virus-like' genetic elements implies that self-splicing introns are among the most ancient genetic entities. Thirdly, reconstructions of the ancestral state of eukaryotic genes suggest that the last common ancestor of extant eukaryotes had an intron

  5. Alternative splicing and trans-splicing events revealed by analysis of the Bombyx mori transcriptome

    OpenAIRE

    Shao, Wei; Zhao, Qiong-Yi; Wang, Xiu-Ye; Xu, Xin-Yan; Tang, Qing; Li, Muwang; Li, Xuan; Xu, Yong-Zhen

    2012-01-01

    Alternative splicing and trans-splicing events have not been systematically studied in the silkworm Bombyx mori. Here, the silkworm transcriptome was analyzed by RNA-seq. The authors identified 320 novel genes, modified 1140 gene models, and found thousands of alternative splicing and 58 trans-splicing events. Studies of three SR proteins show that both their alternative splicing patterns and mRNA products are conserved from insect to human, and one isoform of Srsf6 with a retained intron is ...

  6. Conserved intron positions in ancient protein modules

    Directory of Open Access Journals (Sweden)

    de Roos Albert DG

    2007-02-01

    Full Text Available Abstract Background The timing of the origin of introns is of crucial importance for an understanding of early genome architecture. The Exon theory of genes proposed a role for introns in the formation of multi-exon proteins by exon shuffling and predicts the presence of conserved splice sites in ancient genes. In this study, large-scale analysis of potential conserved splice sites was performed using an intron-exon database (ExInt derived from GenBank. Results A set of conserved intron positions was found by matching identical splice sites sequences from distantly-related eukaryotic kingdoms. Most amino acid sequences with conserved introns were homologous to consensus sequences of functional domains from conserved proteins including kinases, phosphatases, small GTPases, transporters and matrix proteins. These included ancient proteins that originated before the eukaryote-prokaryote split, for instance the catalytic domain of protein phosphatase 2A where a total of eleven conserved introns were found. Using an experimental setup in which the relation between a splice site and the ancientness of its surrounding sequence could be studied, it was found that the presence of an intron was positively correlated to the ancientness of its surrounding sequence. Intron phase conservation was linked to the conservation of the gene sequence and not to the splice site sequence itself. However, no apparent differences in phase distribution were found between introns in conserved versus non-conserved sequences. Conclusion The data confirm an origin of introns deep in the eukaryotic branch and is in concordance with the presence of introns in the first functional protein modules in an 'Exon theory of genes' scenario. A model is proposed in which shuffling of primordial short exonic sequences led to the formation of the first functional protein modules, in line with hypotheses that see the formation of introns integral to the origins of genome evolution

  7. The human decorin gene: Intron-exon organization, discovery of two alternatively spliced exons in the 5[prime] untralsated region, and mapping of the gene to chromosome 12q23

    Energy Technology Data Exchange (ETDEWEB)

    Danielson, K.G.; Fazzio, A.; Cohen, I.; Cannizzaro, L.A.; Eichstetter, I.; Iozzo, R.V. (Thomas Jefferson Univ., Philadelphia, PA (United States))

    1993-01-01

    Decorin is a chondroitin/dermatan sulfate proteoglycan expressed by most vascular and avascular connective tissues and, because of its ability to interact with collagen and growth factors, has been implicated in the control of matrix assembly and cellular growth. To understand the molecular mechanisms involved in regulating its tissue expression, we have isolated a number of genomic clones encoding the complete decorin gene. The human decorin gene spans over 38 kb of continuous DNA sequence and contains eight exons and very large introns, two of which are 5.4 and > 13.2 kb. We have discovered two alternatively spliced leader exons, exons Ia and Ib, in the 5[prime] untranslated region. These exons were identified by cloning and sequencing cDNAs obtained by polymerase chain reaction amplification of a fibroblast cDNA library. Using Northern blotting or reverse transcriptase PCR, we detected the two leader exons in a variety of mRNAs isolated from human cell lines and tissues. Interestingly, sequences highly (74-87%) homologous to exons Ia and lb are found in the 5[prime]untranslated region of avian and bovine decorin, respectively. This high degree of conservation among species suggests regulatory functions for these leader exons. In the 3' untranslated region there are several polyadenylation sites, and at least two of these sites could give rise to the transcripts of [approx]1.6 and [approx]1.9 kb, typically detected in a variety of tissues and cells. Using a genomic clone as the labeled probe and in situ hybridization of human metaphase chromosomes, we have mapped the decorin gene to the discrete region of human chromosome 12q23. This sturdy provides the molecular basis for discerning the transcriptional control of the decorin gene and offers the opportunity to investigate genetic disorders linked to this important human gene. 57 refs., 11 figs., 3 tabs.

  8. Context-dependent splicing regulation: Exon definition, co-occurring motif pairs and tissue specificity

    OpenAIRE

    Ke, Shengdong; CHASIN, LAWRENCE A.

    2011-01-01

    Splicing is a crucial process in gene expression in higher organisms because: (1) most vertebrate genes contain introns; and (2) alternative splicing is primarily responsible for increasing proteomic complexity and functional diversity. Intron definition, the coordination across an intron, is a mandatory step in the splicing process. However, exon definition, the coordination across an exon, is also thought to be required for the splicing of most vertebrate exons. Recent investigations of exo...

  9. Analysis of ribosomal protein gene structures: implications for intron evolution.

    Directory of Open Access Journals (Sweden)

    2006-03-01

    Full Text Available Many spliceosomal introns exist in the eukaryotic nuclear genome. Despite much research, the evolution of spliceosomal introns remains poorly understood. In this paper, we tried to gain insights into intron evolution from a novel perspective by comparing the gene structures of cytoplasmic ribosomal proteins (CRPs and mitochondrial ribosomal proteins (MRPs, which are held to be of archaeal and bacterial origin, respectively. We analyzed 25 homologous pairs of CRP and MRP genes that together had a total of 527 intron positions. We found that all 12 of the intron positions shared by CRP and MRP genes resulted from parallel intron gains and none could be considered to be "conserved," i.e., descendants of the same ancestor. This was supported further by the high frequency of proto-splice sites at these shared positions; proto-splice sites are proposed to be sites for intron insertion. Although we could not definitively disprove that spliceosomal introns were already present in the last universal common ancestor, our results lend more support to the idea that introns were gained late. At least, our results show that MRP genes were intronless at the time of endosymbiosis. The parallel intron gains between CRP and MRP genes accounted for 2.3% of total intron positions, which should provide a reliable estimate for future inferences of intron evolution.

  10. Circularization pathway of a bacterial group II intron.

    Science.gov (United States)

    Monat, Caroline; Cousineau, Benoit

    2016-02-29

    Group II introns are large RNA enzymes that can excise as lariats, circles or in a linear form through branching, circularization or hydrolysis, respectively. Branching is by far the main and most studied splicing pathway while circularization was mostly overlooked. We previously showed that removal of the branch point A residue from Ll.LtrB, the group II intron from Lactococcus lactis, exclusively leads to circularization. However, the majority of the released intron circles harbored an additional C residue of unknown origin at the splice junction. Here, we exploited the Ll.LtrB-ΔA mutant to study the circularization pathway of bacterial group II introns in vivo. We demonstrated that the non-encoded C residue, present at the intron circle splice junction, corresponds to the first nt of exon 2. Intron circularization intermediates, harboring the first 2 or 3 nts of exon 2, were found to accumulate showing that branch point removal leads to 3' splice site misrecognition. Traces of properly ligated exons were also detected functionally confirming that a small proportion of Ll.LtrB-ΔA circularizes accurately. Overall, our data provide the first detailed molecular analysis of the group II intron circularization pathway and suggests that circularization is a conserved splicing pathway in bacteria. PMID:26673697

  11. Social networking between mobile introns and their host genes

    OpenAIRE

    Stoddard, Barry; Belfort, Marlene

    2010-01-01

    Homing endonucleases have long been known as the orchestrators of intron mobility. However, the extent of their influence on the intron and its genetic and cellular environment is still being elucidated. The accompanying paper emphasizes the importance of temporal control of endonuclease expression on splicing, expression of the host gene and cellular metabolism, while it raises questions to guide future inquiry.

  12. A generic intron increases gene expression in transgenic mice.

    OpenAIRE

    Choi, T; Huang, M; Gorman, C; Jaenisch, R

    1991-01-01

    To investigate the role of splicing in the regulation of gene expression, we have generated transgenic mice carrying the human histone H4 promoter linked to the bacterial gene for chloramphenicol acetyltransferase (CAT), with or without a heterologous intron in the transcription unit. We found that CAT activity is 5- to 300-fold higher when the transgene incorporates a hybrid intron than with an analogous transgene precisely deleted for the intervening sequences. This hybrid intron, consistin...

  13. Recursive splicing in long vertebrate genes.

    Science.gov (United States)

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

    2015-05-21

    It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an 'RS-exon' that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5' splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5' splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons. PMID:25970246

  14. Split-Intron Retroviral Vectors: Enhanced Expression with Improved Safety

    OpenAIRE

    Ismail, Said I.; Kingsman, Susan M.; Kingsman, Alan J.; Uden, Mark

    2000-01-01

    The inclusion of retrovirus-derived introns within retrovirus-based expression vectors leads to a fraction of the resulting transcripts being spliced. Such splicing has been shown to markedly improve expression (W. J. Krall et al., Gene Ther. 3:37–48, 1996). One way to improve upon this still further might involve the use of more efficient introns instead of those from the provirus. Currently, however, incorporation of such introns remains self-defeating since they are removed in the nucleus ...

  15. SERRATE is required for intron suppression of RNA silencing in Arabidopsis

    OpenAIRE

    Christie, Michael; Carroll, Bernard J.

    2011-01-01

    Transposons and viruses are generally devoid of introns and are prime targets for small interfering RNAs (siRNAs) and RNA silencing. Conversely, endogenous genes often contain introns and are not usually subjected to post-transcriptional gene silencing by siRNAs. In a recent study, we reported that efficient intron splicing directly suppresses siRNA biogenesis and RNA silencing of a Green Fluorescence Protein (GFP) transgene. Splicing-mediated suppression of GFP silencing was dependent on ABH...

  16. Intron gain by tandem genomic duplication: a novel case in a potato gene encoding RNA-dependent RNA polymerase.

    Science.gov (United States)

    Ma, Ming-Yue; Lan, Xin-Ran; Niu, Deng-Ke

    2016-01-01

    The origin and subsequent accumulation of spliceosomal introns are prominent events in the evolution of eukaryotic gene structure. However, the mechanisms underlying intron gain remain unclear because there are few proven cases of recently gained introns. In an RNA-dependent RNA polymerase (RdRp) gene, we found that a tandem duplication occurred after the divergence of potato and its wild relatives among other Solanum plants. The duplicated sequence crosses the intron-exon boundary of the first intron and the second exon. A new intron was detected at this duplicated region, and it includes a small previously exonic segment of the upstream copy of the duplicated sequence and the intronic segment of the downstream copy of the duplicated sequence. The donor site of this new intron was directly obtained from the small previously exonic segment. Most of the splicing signals were inherited directly from the parental intron/exon structure, including a putative branch site, the polypyrimidine tract, the 3' splicing site, two putative exonic splicing enhancers, and the GC contents differed between the intron and exon. In the widely cited model of intron gain by tandem genomic duplication, the duplication of an AGGT-containing exonic segment provides the GT and AG splicing sites for the new intron. Our results illustrate that the tandem duplication model of intron gain should be diverse in terms of obtaining the proper splicing signals. PMID:27547574

  17. Functional and evolutionary analysis of alternatively spliced genes is consistent with an early eukaryotic origin of alternative splicing

    Directory of Open Access Journals (Sweden)

    Penny David

    2007-10-01

    Full Text Available Abstract Background 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, and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional classes, cellular locations, intron/exon structures and evolutionary origins. Results For each species, we find that genes from most functional categories are alternatively spliced. Ancient genes (shared between animals, fungi and plants show high levels of alternative splicing. Genes with products expressed in the nucleus or plasma membrane are generally more alternatively spliced while those expressed in extracellular location show less alternative splicing. We find a clear correspondence between incidence of alternative splicing and intron number per gene both within and between genomes. In general, we find several similarities in patterns of alternative splicing across these diverse eukaryotes. Conclusion Along with previous studies indicating intron-rich genes with weak intron boundary consensus and complex spliceosomes in ancestral organisms, our results suggest that at least a simple form of alternative splicing may already have been present in the unicellular ancestor of plants, fungi and animals. A role for alternative splicing in the evolution of multicellularity then would largely have arisen by co-opting the preexisting process.

  18. 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. PMID:25720307

  19. MapSplice: accurate mapping of RNA-seq reads for splice junction discovery.

    Science.gov (United States)

    Wang, Kai; Singh, Darshan; Zeng, Zheng; Coleman, Stephen J; Huang, Yan; Savich, Gleb L; He, Xiaping; Mieczkowski, Piotr; Grimm, Sara A; Perou, Charles M; MacLeod, James N; Chiang, Derek Y; Prins, Jan F; Liu, Jinze

    2010-10-01

    The accurate mapping of reads that span splice junctions is a critical component of all analytic techniques that work with RNA-seq data. We introduce a second generation splice detection algorithm, MapSplice, whose focus is high sensitivity and specificity in the detection of splices as well as CPU and memory efficiency. MapSplice can be applied to both short (<75 bp) and long reads (≥ 75 bp). MapSplice is not dependent on splice site features or intron length, consequently it can detect novel canonical as well as non-canonical splices. MapSplice leverages the quality and diversity of read alignments of a given splice to increase accuracy. We demonstrate that MapSplice achieves higher sensitivity and specificity than TopHat and SpliceMap on a set of simulated RNA-seq data. Experimental studies also support the accuracy of the algorithm. Splice junctions derived from eight breast cancer RNA-seq datasets recapitulated the extensiveness of alternative splicing on a global level as well as the differences between molecular subtypes of breast cancer. These combined results indicate that MapSplice is a highly accurate algorithm for the alignment of RNA-seq reads to splice junctions. Software download URL: http://www.netlab.uky.edu/p/bioinfo/MapSplice. PMID:20802226

  20. The agaricus bisporus cox1 gene: the longest mitochondrial gene and the largest reservoir of mitochondrial group I introns

    OpenAIRE

    Cyril Férandon; Serge Moukha; Philippe Callac; Jean-Pierre Benedetto; Michel Castroviejo; Gérard Barroso

    2010-01-01

    In eukaryotes, introns are located in nuclear and organelle genes from several kingdoms. Large introns (up to 5 kbp) are frequent in mitochondrial genomes of plant and fungi but scarce in Metazoa, even if these organisms are grouped with fungi among the Opisthokonts. Mitochondrial introns are classified in two groups (I and II) according to their RNA secondary structure involved in the intron self-splicing mechanism. Most of these mitochondrial group I introns carry a “Homing Endonuclease Gen...

  1. Isolation and characterization of functional tripartite group II introns using a Tn5-based genetic screen.

    Directory of Open Access Journals (Sweden)

    Christine Ritlop

    Full Text Available BACKGROUND: Group II introns are RNA enzymes that splice themselves from pre-mRNA transcripts. Most bacterial group II introns harbour an open reading frame (ORF, coding for a protein with reverse transcriptase, maturase and occasionally DNA binding and endonuclease activities. Some ORF-containing group II introns were shown to be mobile retroelements that invade new DNA target sites. From an evolutionary perspective, group II introns are hypothesized to be the ancestors of the spliceosome-dependent nuclear introns and the small nuclear RNAs (snRNAs--U1, U2, U4, U5 and U6 that are important functional elements of the spliceosome machinery. The ability of some group II introns fragmented in two or three pieces to assemble and undergo splicing in trans supports the theory that spliceosomal snRNAs evolved from portions of group II introns. METHODOLOGY/PRINCIPAL FINDINGS: We used a transposon-based genetic screen to explore the ability of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis to be fragmented into three pieces in vivo. Trans-splicing tripartite variants of Ll.LtrB were selected using a highly efficient and sensitive trans-splicing/conjugation screen. We report that numerous fragmentation sites located throughout Ll.LtrB support tripartite trans-splicing, showing that this intron is remarkably tolerant to fragmentation. CONCLUSIONS/SIGNIFICANCE: This work unveils the great versatility of group II intron fragments to assemble and accurately trans-splice their flanking exons in vivo. The selected introns represent the first evidence of functional tripartite group II introns in bacteria and provide experimental support for the proposed evolutionary relationship between group II introns and snRNAs.

  2. Exon definition as a potential negative force against intron losses in evolution

    OpenAIRE

    Niu Deng-Ke

    2008-01-01

    Abstract Background Previous studies have indicated that the wide variation in intron density (the number of introns per gene) among different eukaryotes largely reflects varying degrees of intron loss during evolution. The most popular model, which suggests that organisms lose introns through a mechanism in which reverse-transcribed cDNA recombines with the genomic DNA, concerns only one mutational force. Hypothesis Using exons as the units of splicing-site recognition, exon definition const...

  3. Control of Pre-mRNA Splicing by the General Splicing Factors PUF60 and U2AF65

    OpenAIRE

    Hastings, Michelle L.; Eric Allemand; Duelli, Dominik M.; Michael P Myers; Krainer, Adrian R.

    2007-01-01

    Pre-mRNA splicing is a crucial step in gene expression, and accurate recognition of splice sites is an essential part of this process. Splice sites with weak matches to the consensus sequences are common, though it is not clear how such sites are efficiently utilized. Using an in vitro splicing-complementation approach, we identified PUF60 as a factor that promotes splicing of an intron with a weak 3' splice-site. PUF60 has homology to U2AF(65), a general splicing factor that facilitates 3' s...

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

    OpenAIRE

    Manuel Irimia; Jakob Lewin Rukov; Scott William Roy

    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 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 identifie...

  5. Group I intron ribozymes

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2012-01-01

    Group I intron ribozymes constitute one of the main classes of ribozymes and have been a particularly important model in the discovery of key concepts in RNA biology as well as in the development of new methods. Compared to other ribozyme classes, group I intron ribozymes display considerable...... intronic products of these pathways have the potential to integrate into targets and to form various types of circular RNA molecules. Thus, group I intron ribozymes and associated elements found within group I introns is a rich source of biological phenomena. This chapter provides a strategy and protocols...... for initial characterization of new group I intron ribozymes....

  6. Position dependence of the rous sarcoma virus negative regulator of splicing element reflects proximity to a 5' splice site

    International Nuclear Information System (INIS)

    Rous sarcoma virus (RSV) requires incomplete splicing of its viral transcripts to maintain efficient replication. A splicing inhibitor element, the negative regulator of splicing (NRS), is located near the 5' end of the RNA but the significance of this positioning is not known. In a heterologous intron the NRS functions optimally when positioned close to the authentic 5' splice site. This observation led us to investigate the basis of the position dependence. Four explanations were put forth and stressed the role of three major elements involved in splicing, the 3' splice site, the 5' splice site, and the 5' end cap structure. NRS function was unrelated to its position relative to the 3' splice site or the cap structure and appeared to depend on its position relative to the authentic 5' splice site. We conclude that position dependence may reflect distance constraints necessary for competition of the NRS with the authentic 5' splice site for pairing with the 3' splice sites

  7. 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

    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......: 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...... 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 in...

  8. 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

    , and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional...... classes, cellular locations, intron/exon structures and evolutionary origins. RESULTS: For each species, we find that genes from most functional categories are alternatively spliced. Ancient genes (shared between animals, fungi and plants) show high levels of alternative splicing. Genes with products...... expressed in the nucleus or plasma membrane are generally more alternatively spliced while those expressed in extracellular location show less alternative splicing. We find a clear correspondence between incidence of alternative splicing and intron number per gene both within and between genomes. In general...

  9. Regulation of alternative splice site selection by reversible protein phosphorylation

    OpenAIRE

    Novoyatleva, Tatyana

    2007-01-01

    Splicing is the process that removes introns and joins exons from pre-mesenger RNA (pre-mRNA). It is an essential step in pre-mRNA processing that form the mature RNA. Microarray data indicates that approximately 75% of human genes produce transcripts that are alternatively spliced. Alternative splicing is one of the major mechanisms that ultimately generate high number of protein isoforms from a limited number of genes. The proper catalysis and regulation of alternative splice site selection...

  10. Autogenous Regulation of Splicing of the Transcript of a Yeast Ribosomal Protein Gene

    Science.gov (United States)

    Dabeva, Mariana D.; Post-Beittenmiller, Martha A.; Warner, Jonathan R.

    1986-08-01

    The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.

  11. Autogenous regulation of splicing of the transcript of a yeast ribosomal protein gene.

    OpenAIRE

    Dabeva, M. D.; Post-Beittenmiller, M A; Warner, J R

    1986-01-01

    The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Group II intron-based gene targeting reactions in eukaryotes.

    Directory of Open Access Journals (Sweden)

    Marta Mastroianni

    Full Text Available Mobile group II introns insert site-specifically into DNA target sites by a mechanism termed retrohoming in which the excised intron RNA reverse splices into a DNA strand and is reverse transcribed by the intron-encoded protein. Retrohoming is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and excised intron RNA, with target specificity determined largely by base pairing of the intron RNA to the DNA target sequence. This feature enabled the development of mobile group II introns into bacterial gene targeting vectors ("targetrons" with programmable target specificity. Thus far, however, efficient group II intron-based gene targeting reactions have not been demonstrated in eukaryotes.By using a plasmid-based Xenopus laevis oocyte microinjection assay, we show that group II intron RNPs can integrate efficiently into target DNAs in a eukaryotic nucleus, but the reaction is limited by low Mg(2+ concentrations. By supplying additional Mg(2+, site-specific integration occurs in up to 38% of plasmid target sites. The integration products isolated from X. laevis nuclei are sensitive to restriction enzymes specific for double-stranded DNA, indicating second-strand synthesis via host enzymes. We also show that group II intron RNPs containing either lariat or linear intron RNA can introduce a double-strand break into a plasmid target site, thereby stimulating homologous recombination with a co-transformed DNA fragment at frequencies up to 4.8% of target sites. Chromatinization of the target DNA inhibits both types of targeting reactions, presumably by impeding RNP access. However, by using similar RNP microinjection methods, we show efficient Mg(2+-dependent group II intron integration into plasmid target sites in zebrafish (Danio rerio embryos and into plasmid and chromosomal target sites in Drosophila melanogster embryos, indicating that DNA replication can mitigate effects of chromatinization.Our results provide an

  14. Invasion of protein coding genes by green algal ribosomal group I introns.

    Science.gov (United States)

    McManus, Hilary A; Lewis, Louise A; Fučíková, Karolina; Haugen, Peik

    2012-01-01

    The spread of group I introns depends on their association with intron-encoded homing endonucleases. Introns that encode functional homing endonuclease genes (HEGs) are highly invasive, whereas introns that only encode the group I ribozyme responsible for self-splicing are generally stably inherited (i.e., vertical inheritance). A number of recent case studies have provided new knowledge on the evolution of group I introns, however, there are still large gaps in understanding of their distribution on the tree of life, and how they have spread into new hosts and genic sites. During a larger phylogenetic survey of chlorophyceaen green algae, we found that 23 isolates contain at least one group I intron in the rbcL chloroplast gene. Structural analyses show that the introns belong to one of two intron lineages, group IA2 intron-HEG (GIY-YIG family) elements inserted after position 462 in the rbcL gene, and group IA1 introns inserted after position 699. The latter intron type sometimes encodes HNH homing endonucleases. The distribution of introns was analyzed on an exon phylogeny and patterns were recovered that are consistent with vertical inheritance and possible horizontal transfer. The rbcL 462 introns are thus far reported only within the Volvocales, Hydrodictyaceae and Bracteacoccus, and closely related isolates of algae differ in the presence of rbcL introns. Phylogenetic analysis of the intron conserved regions indicates that the rbcL699 and rbcL462 introns have distinct evolutionary origins. The rbcL699 introns were likely derived from ribosomal RNA L2449 introns, whereas the rbcL462 introns form a close relationship with psbA introns. PMID:22056605

  15. EVOLUTION OF SR PROTEIN AND HnRNP SPLICING REGULATORY FACTORS

    OpenAIRE

    Busch, A.; Hertel, KJ

    2011-01-01

    The splicing of pre-mRNAs is an essential step of gene expression in eukaryotes. Introns are removed from split genes through the activities of the spliceosome, a large ribonuclear machine that is conserved throughout the eukaryotic lineage. While unicellular eukaryotes are characterized by less complex splicing, pre-mRNA splicing of multicellular organisms is often associated with extensive alternative splicing that significantly enriches their proteome. The alternative selection of splice s...

  16. Nascent-seq indicates widespread cotranscriptional pre-mRNA splicing in Drosophila

    OpenAIRE

    Khodor, Yevgenia L.; Rodriguez, Joseph; Abruzzi, Katharine C.; Tang, Chih-Hang Anthony; Marr, Michael T.; Rosbash, Michael

    2011-01-01

    Cotranscriptional splicing, in which mRNA is spliced as it is being transcribed, is thought to be necessary for proper gene regulation of many genes in eukaryotic cells. While studies have shown that splicing takes place cotranscriptionally in yeast, in higher eukaryotes, where genes contain multiple introns with widespread alternative splicing, the question of whether cotranscriptional splicing is a general phenomenon remains. Khodor et al. investigated what fractions of genes are cotranscri...

  17. Split-intron retroviral vectors: enhanced expression with improved safety.

    Science.gov (United States)

    Ismail, S I; Kingsman, S M; Kingsman, A J; Uden, M

    2000-03-01

    The inclusion of retrovirus-derived introns within retrovirus-based expression vectors leads to a fraction of the resulting transcripts being spliced. Such splicing has been shown to markedly improve expression (W. J. Krall et al., Gene Ther. 3:37-48, 1996). One way to improve upon this still further might involve the use of more efficient introns instead of those from the provirus. Currently, however, incorporation of such introns remains self-defeating since they are removed in the nucleus of the producer cell. In the past, elaborate ways to overcome this problem have included the use of alphaviruses to make the vector transcripts within the cytoplasm, thus avoiding the nuclear splicing machinery during vector production (K. J. Li and H. Garoff, Proc. Natl. Acad. Sci. USA 95:3650-3654, 1998). We now present a novel design for the inclusion of introns within a retroviral vector. In essence, this is achieved by exploiting the retroviral replication process to copy not only the U3 promoter but also a synthetic splice donor to the 5'-long-terminal-repeat position during reverse transcription. Once copied, synthesized transcripts then contain a splice donor at their 5' end capable of interacting with a consensus splice acceptor engineered downstream of the packaging signal. Upon transduction, we demonstrate these vectors to produce enhanced expression from near fully spliced (and thus packaging signal minus) transcripts. The unique design of these high titer and high-expression retroviral vectors may be of use in a number of gene therapy applications. PMID:10666267

  18. Functional comparison of three transformer gene introns regulating conditional female lethality

    Science.gov (United States)

    The trasformer gene plays a critical role in the sex determination pathways of many insects. We cloned two transformer gene introns from Anastrepha suspensa, the Caribbean fruit fly. These introns have sequences that putatively have a role in sex-specific splicing patterns that affect sex determinat...

  19. Introns in the genome of bacteriophage T4

    International Nuclear Information System (INIS)

    RNA from T4-infected cells yields multiple end-labeled species when incubated with [α-32P]GTP under self-splicing conditions. One of these corresponds to the previously characterized intron from the T4 td gene and, as shown in this work, the others represent additional group I introns in T4. Two loci distinct from the td gene were found to hybridize to the mixed GTP-labeled T4 RNA probe. These were mapped to the unlinked genes nrdB and sunY. Cloned DNA from the nrdB region that contained the intron was shown to generate characteristic group I splice products with RNA synthesized in vivo or in vitro. The splice junction of the nrdB gene was determined and the nature of the RNA reaction products characterized. In vivo expression of the nrdB gene and the open reading frame within the intron was studied using in-frame lacZ fusions and primer extension analyses. The data suggest that expression of the intron open reading frame is highly regulated during T4 infection. Possible regulatory mechanisms are discussed

  20. The ability to form full-length intron RNA circles is a general property of nuclear group I introns

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Fiskaa, Tonje; Birgisdottir, Asa Birna; Haugen, Peik; Einvik, Christer; Johansen, Steinar

    2003-01-01

    at the expense of the host. The circularization pathway has distinct structural requirements that differ from those of splicing and appears to be specifically suppressed in vivo. The ability to form full-length circles is found in all types of nuclear group I introns, including those from the...

  1. Minor class splicing shapes the zebrafish transcriptome during development

    DEFF Research Database (Denmark)

    Markmiller, Sebastian; Cloonan, Nicole; Lardelli, Rea M;

    2014-01-01

    Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities...

  2. Phosphorylation-Mediated Regulation of Alternative Splicing in Cancer

    OpenAIRE

    Chiara Naro; Claudio Sette

    2013-01-01

    Alternative splicing (AS) is one of the key processes involved in the regulation of gene expression in eukaryotic cells. AS catalyzes the removal of intronic sequences and the joining of selected exons, thus ensuring the correct processing of the primary transcript into the mature mRNA. The combinatorial nature of AS allows a great expansion of the genome coding potential, as multiple splice-variants encoding for different proteins may arise from a single gene. Splicing is mediated by a large...

  3. Embracing the complexity of pre-mRNA splicing

    Institute of Scientific and Technical Information of China (English)

    Peter J Shepard; Klemens J Hertel

    2010-01-01

    @@ Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome, which catalyzes the removal of non-coding intronic sequences to assemble exons into mature mRNAs prior to export and translation.Defects in splicing lead to many human genetic diseases [1], and splicing mutations in a number of genes involved in growth control have been implicated in multiple types of cancer.

  4. Size and position of intervening sequences are critical for the splicing efficiency of pre-mRNA in the yeast Saccharomyces cerevisiae.

    OpenAIRE

    Klinz, F. J.; Gallwitz, D

    1985-01-01

    The size of the 309 bp actin gene intron of the yeast Saccharomyces cerevisiae was enlarged by inserting DNA fragments of different lengths and sequence. Enlarging the intron above 551 bp, the largest known yeast intron, led to a decrease in splicing efficiency. The effect on transcript splicing was dependent on the length of the inserted fragments rather than their sequence. It was also observed that insertion of the actin gene intron into different regions of the normally unsplit yeast YP2 ...

  5. Proximity-dependent and proximity-independent trans-splicing in mammalian cells

    OpenAIRE

    Viles, Kristi D.; Sullenger, Bruce A

    2008-01-01

    Most human pre-mRNAs are cis-spliced, removing introns and joining flanking exons of the same RNA molecule. However, splicing of exons present on separate pre-mRNA molecules can also occur. This trans-splicing reaction can be exploited by pre-trans-splicing molecules (PTMs), which are incapable of cis-splicing. PTM-mediated trans-splicing has been utilized to repair mutant RNAs as a novel approach to gene therapy. Herein we explore how the site of PTM expression influences trans-splicing acti...

  6. 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.

  7. 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.

  8. Adenosine to Inosine editing frequency controlled by splicing efficiency.

    Science.gov (United States)

    Licht, Konstantin; Kapoor, Utkarsh; Mayrhofer, Elisa; Jantsch, Michael F

    2016-07-27

    Alternative splicing and adenosine to inosine (A to I) RNA-editing are major factors leading to co- and post-transcriptional modification of genetic information. Both, A to I editing and splicing occur in the nucleus. As editing sites are frequently defined by exon-intron basepairing, mRNA splicing efficiency should affect editing levels. Moreover, splicing rates affect nuclear retention and will therefore also influence the exposure of pre-mRNAs to the editing-competent nuclear environment. Here, we systematically test the influence of splice rates on RNA-editing using reporter genes but also endogenous substrates. We demonstrate for the first time that the extent of editing is controlled by splicing kinetics when editing is guided by intronic elements. In contrast, editing sites that are exclusively defined by exonic structures are almost unaffected by the splicing efficiency of nearby introns. In addition, we show that editing levels in pre- and mature mRNAs do not match. This phenomenon can in part be explained by the editing state of an RNA influencing its splicing rate but also by the binding of the editing enzyme ADAR that interferes with splicing. PMID:27112566

  9. Molecular characterization of a new member of the lariat capping twin-ribozyme introns

    DEFF Research Database (Denmark)

    Tang, Yunjia; Nielsen, Henrik; Masquida, Benoît;

    2014-01-01

    BACKGROUND: Twin-ribozyme introns represent a complex class of mobile group I introns that harbour a lariat capping (LC) ribozyme and a homing endonuclease gene embedded in a conventional self-splicing group I ribozyme (GIR2). Twin-ribozyme introns have so far been confined to nucleolar DNA in...... Naegleria amoeboflagellates and the myxomycete Didymium iridis. RESULTS: We characterize structural organization, catalytic properties and molecular evolution of a new twin-ribozyme intron in Allovahlkampfia (Heterolobosea). The intron contains two ribozyme domains with different functions in ribosomal RNA...... efficient self-cleaving ribozyme that generates a small 2',5' lariat cap at the 5' end of the homing endonuclease mRNA, and thus contributes to intron mobility. CONCLUSIONS: The discovery of a twin-ribozyme intron in a member of Heterolobosea expands the distribution pattern of LC ribozymes. We identify a...

  10. U12 type introns were lost at multiple occasions during evolution

    Directory of Open Access Journals (Sweden)

    Bartschat Sebastian

    2010-02-01

    Full Text Available Abstract Background Two categories of introns are known, a common U2 type and a rare U12 type. These two types of introns are removed by distinct spliceosomes. The phylogenetic distribution of spliceosomal RNAs that are characteristic of the U12 spliceosome, i.e. the U11, U12, U4atac and U6atac RNAs, suggest that U12 spliceosomes were lost in many phylogenetic groups. We have now examined the distribution of U2 and U12 introns in many of these groups. Results U2 and U12 introns were predicted by making use of available EST and genomic sequences. The results show that in species or branches where U12 spliceosomal components are missing, also U12 type of introns are lacking. Examples are the choanoflagellate Monosiga brevicollis, Entamoeba histolytica, green algae, diatoms, and the fungal lineage Basidiomycota. Furthermore, whereas U12 splicing does not occur in Caenorhabditis elegans, U12 introns as well as U12 snRNAs are present in Trichinella spiralis, which is deeply branching in the nematode tree. A comparison of homologous genes in T. spiralis and C. elegans revealed different mechanisms whereby U12 introns were lost. Conclusions The phylogenetic distribution of U12 introns and spliceosomal RNAs give further support to an early origin of U12 dependent splicing. In addition, this distribution identifies a large number of instances during eukaryotic evolution where such splicing was lost.

  11. 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

  12. Bacterial Group II Introns in a Deep-Sea Hydrothermal Vent Environment†

    OpenAIRE

    Podar, Mircea; Mullineaux, Lauren; Huang, Hon-Ren; Perlman, Philip S.; Sogin, Mitchell L.

    2002-01-01

    Group II introns are catalytic RNAs and mobile retrotransposable elements known to be present in the genomes of some nonmarine bacteria and eukaryotic organelles. Here we report the discovery of group II introns in a bacterial mat sample collected from a deep-sea hydrothermal vent near 9°N on the East Pacific Rise. One of the introns was shown to self-splice in vitro. This is the first example of marine bacterial introns from molecular population structure studies of microorganisms that live ...

  13. Exon definition may facilitate splice site selection in RNAs with multiple exons.

    OpenAIRE

    Robberson, B L; Cote, G J; Berget, S M

    1990-01-01

    Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs...

  14. Inducible Expression and Splicing of Candida Group Ⅰ Ribozyme in E.coli

    Institute of Scientific and Technical Information of China (English)

    SHANG Yuan; WANG Chen; ZHANG Yi

    2005-01-01

    The Ca. LSU intron flanking a 129 bp exon upstream and a 100 bp exon downstream was inserted into the lacZ gene on pRS426 to transform E. coli. Northern blot analysis and RT-PCR showed that splicing of Ca. LSU in E.coli is efficient upon inducible expression of the precursor RNA. In contrast, co-transcriptional self-splicing of the intron in vitro is much less active. Therefore, this E. coli splicing system can be used as a better model to investigate the effect of the ribozyme inhibitors on Ca. LSU splicing in living cell. We examined the effects of neomycin sulfate and pentamidine on Ca. LSU splicing in E. coli, and found that these drugs does-dependently inhibit the intron splicing.However, heomycin is more potent than pentamidine in this action.

  15. Normal and abnormal mechanisms of gene splicing and relevance to inherited skin diseases

    OpenAIRE

    Wessagowit, Vesarat; Nalla, Vijay K.; Rogan, Peter K; McGrath, John A

    2005-01-01

    The process of excising introns from pre-mRNA complexes is directed by specific genomic DNA sequences at intron—exon borders known as splice sites. These regions contain well-conserved motifs which allow the splicing process to proceed in a regulated and structured manner. However, as well as conventional splicing, several genes have the inherent capacity to undergo alternative splicing, thus allowing synthesis of multiple gene transcripts, perhaps with different functional properties. Within...

  16. Multiple gains of spliceosomal introns in a superfamily of vertebrate protease inhibitor genes

    Directory of Open Access Journals (Sweden)

    Frese Marc-André

    2009-08-01

    Full Text Available Abstract Background Intron gains reportedly are very rare during evolution of vertebrates, and the mechanisms underlying their creation are largely unknown. Previous investigations have shown that, during metazoan radiation, the exon-intron patterns of serpin superfamily genes were subject to massive changes, in contrast to many other genes. Results Here we investigated intron dynamics in the serpin superfamily in lineages pre- and postdating the split of vertebrates. Multiple intron gains were detected in a group of ray-finned fishes, once the canonical groups of vertebrate serpins had been established. In two genes, co-occurrence of non-standard introns was observed, implying that intron gains in vertebrates may even happen concomitantly or in a rapidly consecutive manner. DNA breakage/repair processes associated with genome compaction are introduced as a novel factor potentially favoring intron gain, since all non-canonical introns were found in a lineage of ray-finned fishes that experienced genomic downsizing. Conclusion Multiple intron acquisitions were identified in serpin genes of a lineage of ray-finned fishes, but not in any other vertebrates, suggesting that insertion rates for introns may be episodically increased. The co-occurrence of non-standard introns within the same gene discloses the possibility that introns may be gained simultaneously. The sequences flanking the intron insertion points correspond to the proto-splice site consensus sequence MAG↑N, previously proposed to serve as intron insertion site. The association of intron gains in the serpin superfamily with a group of fishes that underwent genome compaction may indicate that DNA breakage/repair processes might foster intron birth.

  17. Characterization of rbcL group IA introns from two colonial volvocalean species (Chlorophyceae).

    Science.gov (United States)

    Nozaki, H; Ohta, N; Yamada, T; Takano, H

    1998-05-01

    Group I introns were reported for the first time in the large subunit of Rubisco (rbcL) genes, using two colonial green algae, Pleodorina californica and Gonium multicoccum (Volvocales). The rbcL gene of P. californica contained an intron (PIC intron) of 1320 bp harboring an open reading frame (ORF). The G. multicoccum rbcL gene had two ORF-lacking introns of 549 (GM1 intron) and 295 (GM2 intron) base pairs. Based on the conserved nucleotide sequences of the secondary structure, the PIC and GM1 introns were assigned to group IA2 whereas the GM2 intron belonged to group IA1. Southern hybridization analyses of nuclear and chloroplast DNAs indicated that such intron-containing rbcL genes are located in the chloroplast genome. Sequencing RNAs from the two algae revealed that these introns are spliced out during mRNA maturation. In addition, the PIC and GM1 introns were inserted in the same position of the rbcL exons, and phylogenetic analysis of group IA introns indicated a close phylogenetic relationship between the PIC and GM1 introns within the lineage of bacteriophage group IA2 introns. However, P. californica and G. multicoccum occupy distinct clades in the phylogenetic trees of the colonial Volvocales, and the majority of other colonial volvocalean species do not have such introns in the rbcL genes. Therefore, these introns might have been recently inserted in the rbcL genes independently by horizontal transmission by viruses or bacteriophage. PMID:9620266

  18. Critical association of ncRNA with introns

    OpenAIRE

    Rearick, David; Prakash, Ashwin; McSweeny, Andrew; Shepard, Samuel S.; Fedorova, Larisa; Fedorov, Alexei

    2010-01-01

    It has been widely acknowledged that non-coding RNAs are master-regulators of genomic functions. However, the significance of the presence of ncRNA within introns has not received proper attention. ncRNA within introns are commonly produced through the post-splicing process and are specific signals of gene transcription events, impacting many other genes and modulating their expression. This study, along with the following discussion, details the association of thousands of ncRNAs—snoRNA, miR...

  19. Complex group-I introns in nuclear SSU rDNA of red and green algae: evidence of homing-endonuclease pseudogenes in the Bangiophyceae

    DEFF Research Database (Denmark)

    Haugen, P; Huss, V A; Nielsen, Henrik;

    1999-01-01

    The green alga Scenedesmus pupukensis and the red alga Porphyra spiralis contain large group-IC1 introns in their nuclear small subunit ribosomal RNA genes due to the presence of open reading frames at the 5' end of the introns. The putative 555 amino-acid Scenedesmus-encoded protein harbors a...... sequence motif resembling the bacterial S9 ribosomal proteins. The Porphyra intron self-splices in vitro, and generates both ligated exons and a full-length intron RNA circle. The Porphyra intron has an unusual structural organization by encoding a potential 149 amino-acid homing-endonuclease-like protein...... functions of nuclear group-I intron proteins....

  20. BRR2a Affects Flowering Time via FLC Splicing.

    Science.gov (United States)

    Mahrez, Walid; Shin, Juhyun; Muñoz-Viana, Rafael; Figueiredo, Duarte D; Trejo-Arellano, Minerva S; Exner, Vivien; Siretskiy, Alexey; Gruissem, Wilhelm; Köhler, Claudia; Hennig, Lars

    2016-04-01

    Several pathways control time to flowering in Arabidopsis thaliana through transcriptional and posttranscriptional gene regulation. In recent years, mRNA processing has gained interest as a critical regulator of flowering time control in plants. However, the molecular mechanisms linking RNA splicing to flowering time are not well understood. In a screen for Arabidopsis early flowering mutants we identified an allele of BRR2a. BRR2 proteins are components of the spliceosome and highly conserved in eukaryotes. Arabidopsis BRR2a is ubiquitously expressed in all analyzed tissues and involved in the processing of flowering time gene transcripts, most notably FLC. A missense mutation of threonine 895 in BRR2a caused defects in FLC splicing and greatly reduced FLC transcript levels. Reduced FLC expression increased transcription of FT and SOC1 leading to early flowering in both short and long days. Genome-wide experiments established that only a small set of introns was not correctly spliced in the brr2a mutant. Compared to control introns, retained introns were often shorter and GC-poor, had low H3K4me1 and CG methylation levels, and were often derived from genes with a high-H3K27me3-low-H3K36me3 signature. We propose that BRR2a is specifically needed for efficient splicing of a subset of introns characterized by a combination of factors including intron size, sequence and chromatin, and that FLC is most sensitive to splicing defects. PMID:27100965

  1. Phylogenetic distribution of intron positions in alpha-amylase genes of bilateria suggests numerous gains and losses.

    Directory of Open Access Journals (Sweden)

    Jean-Luc Da Lage

    Full Text Available Most eukaryotes have at least some genes interrupted by introns. While it is well accepted that introns were already present at moderate density in the last eukaryote common ancestor, the conspicuous diversity of intron density among genomes suggests a complex evolutionary history, with marked differences between phyla. The question of the rates of intron gains and loss in the course of evolution and factors influencing them remains controversial. We have investigated a single gene family, alpha-amylase, in 55 species covering a variety of animal phyla. Comparison of intron positions across phyla suggests a complex history, with a likely ancestral intronless gene undergoing frequent intron loss and gain, leading to extant intron/exon structures that are highly variable, even among species from the same phylum. Because introns are known to play no regulatory role in this gene and there is no alternative splicing, the structural differences may be interpreted more easily: intron positions, sizes, losses or gains may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors, such as life cycle and population size. We have shown that intron losses outnumbered gains in recent periods, but that "resets" of intron positions occurred at the origin of several phyla, including vertebrates. Rates of gain and loss appear to be positively correlated. No phase preference was found. We also found evidence for parallel gains and for intron sliding. Presence of introns at given positions was correlated to a strong protosplice consensus sequence AG/G, which was much weaker in the absence of intron. In contrast, recent intron insertions were not associated with a specific sequence. In animal Amy genes, population size and generation time seem to have played only minor roles in shaping gene structures.

  2. SF3B1 Association with Chromatin Determines Splicing Outcomes

    Directory of Open Access Journals (Sweden)

    Nir Kfir

    2015-04-01

    Full Text Available Much remains unknown concerning the mechanism by which the splicing machinery pinpoints short exons within intronic sequences and how splicing factors are directed to their pre-mRNA targets. One probable explanation lies in differences in chromatin organization between exons and introns. Proteomic, co-immunoprecipitation, and sedimentation analyses described here indicate that SF3B1, an essential splicing component of the U2 snRNP complex, is strongly associated with nucleosomes. ChIP-seq and RNA-seq analyses reveal that SF3B1 specifically binds nucleosomes located at exonic positions. SF3B1 binding is enriched at nucleosomes positioned over short exons flanked by long introns that are also characterized by differential GC content between exons and introns. Disruption of SF3B1 binding to such nucleosomes affects splicing of these exons similarly to SF3B1 knockdown. Our findings suggest that the association of SF3B1 with nucleosomes is functionally important for splice-site recognition and that SF3B1 conveys splicing-relevant information embedded in chromatin structure.

  3. An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum

    Science.gov (United States)

    Burggraf, S.; Larsen, N.; Woese, C. R.; Stetter, K. O.

    1993-01-01

    The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

  4. Cyanobacterial ribosomal RNA genes with multiple, endonuclease-encoding group I introns

    Directory of Open Access Journals (Sweden)

    Turner Seán

    2007-09-01

    Full Text Available Abstract Background Group I introns are one of the four major classes of introns as defined by their distinct splicing mechanisms. Because they catalyze their own removal from precursor transcripts, group I introns are referred to as autocatalytic introns. Group I introns are common in fungal and protist nuclear ribosomal RNA genes and in organellar genomes. In contrast, they are rare in all other organisms and genomes, including bacteria. Results Here we report five group I introns, each containing a LAGLIDADG homing endonuclease gene (HEG, in large subunit (LSU rRNA genes of cyanobacteria. Three of the introns are located in the LSU gene of Synechococcus sp. C9, and the other two are in the LSU gene of Synechococcus lividus strain C1. Phylogenetic analyses show that these introns and their HEGs are closely related to introns and HEGs located at homologous insertion sites in organellar and bacterial rDNA genes. We also present a compilation of group I introns with homing endonuclease genes in bacteria. Conclusion We have discovered multiple HEG-containing group I introns in a single bacterial gene. To our knowledge, these are the first cases of multiple group I introns in the same bacterial gene (multiple group I introns have been reported in at least one phage gene and one prophage gene. The HEGs each contain one copy of the LAGLIDADG motif and presumably function as homodimers. Phylogenetic analysis, in conjunction with their patchy taxonomic distribution, suggests that these intron-HEG elements have been transferred horizontally among organelles and bacteria. However, the mode of transfer and the nature of the biological connections among the intron-containing organisms are unknown.

  5. Introns: The Functional Benefits of Introns in Genomes.

    Science.gov (United States)

    Jo, Bong-Seok; Choi, Sun Shim

    2015-12-01

    The intron has been a big biological mystery since it was first discovered in several aspects. First, all of the completely sequenced eukaryotes harbor introns in the genomic structure, whereas no prokaryotes identified so far carry introns. Second, the amount of total introns varies in different species. Third, the length and number of introns vary in different genes, even within the same species genome. Fourth, all introns are copied into RNAs by transcription and DNAs by replication processes, but intron sequences do not participate in protein-coding sequences. The existence of introns in the genome should be a burden to some cells, because cells have to consume a great deal of energy to copy and excise them exactly at the correct positions with the help of complicated spliceosomal machineries. The existence throughout the long evolutionary history is explained, only if selective advantages of carrying introns are assumed to be given to cells to overcome the negative effect of introns. In that regard, we summarize previous research about the functional roles or benefits of introns. Additionally, several other studies strongly suggesting that introns should not be junk will be introduced. PMID:26865841

  6. 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.

  7. Altered PLP1 splicing causes hypomyelination of early myelinating structures

    Science.gov (United States)

    Kevelam, Sietske H; Taube, Jennifer R; van Spaendonk, Rosalina M L; Bertini, Enrico; Sperle, Karen; Tarnopolsky, Mark; Tonduti, Davide; Valente, Enza Maria; Travaglini, Lorena; Sistermans, Erik A; Bernard, Geneviève; Catsman-Berrevoets, Coriene E; van Karnebeek, Clara D M; Østergaard, John R; Friederich, Richard L; Fawzi Elsaid, Mahmoud; Schieving, Jolanda H; Tarailo-Graovac, Maja; Orcesi, Simona; Steenweg, Marjan E; van Berkel, Carola G M; Waisfisz, Quinten; Abbink, Truus E M; van der Knaap, Marjo S; Hobson, Grace M; Wolf, Nicole I

    2015-01-01

    Objective The objective of this study was to investigate the genetic etiology of the X-linked disorder “Hypomyelination of Early Myelinating Structures” (HEMS). Methods We included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search 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 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 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 in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio. Interpretation Brain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus–Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing. PMID:26125040

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

    Science.gov (United States)

    Plotch, Stephen J.; Krug, Robert M.

    1986-08-01

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

  10. 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.

  11. An intron in the thymidylate synthase gene of Bacillus bacteriophage beta 22: evidence for independent evolution of a gene, its group I intron, and the intron open reading frame.

    Science.gov (United States)

    Bechhofer, D H; Hue, K K; Shub, D A

    1994-11-22

    The thymidylate synthase gene (thy) (EC 2.1.1.45) of Bacillus subtilis bacteriophage beta 22 has a self-splicing, group I intron inserted into a highly conserved region of the coding sequence. The intron is very similar to one that is inserted 21 bp further downstream in the homologous thymidylate synthase gene (td) of Escherichia coli bacteriophage T4. In contrast, the amino acid sequences of the bacteriophage thymidylate synthases are highly divergent. The beta 22 intron has a fragmentary open reading frame (ORF) that encodes a putative helix-turn-helix DNA-binding motif, similar to one at the carboxyl terminus of the homing endonuclease (I-TevI) encoded by the T4 td intron. The td ORF and the thy ORF fragments are inserted into different regions of their respective intron structures. These results suggest that the thymidylate synthase genes, their introns, and their respective intron-ORFs all have separate evolutionary histories and that the acquisition of the intron could not have occurred by a simple homing event. PMID:7972121

  12. 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. PMID:20512402

  13. 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.

  14. Purifying Selection on Exonic Splice Enhancers in Intronless Genes.

    Science.gov (United States)

    Savisaar, Rosina; Hurst, Laurence D

    2016-06-01

    Exonic splice enhancers (ESEs) are short nucleotide motifs, enriched near exon ends, that enhance the recognition of the splice site and thus promote splicing. Are intronless genes under selection to avoid these motifs so as not to attract the splicing machinery to an mRNA that should not be spliced, thereby preventing the production of an aberrant transcript? Consistent with this possibility, we find that ESEs in putative recent retrocopies are at a higher density and evolving faster than those in other intronless genes, suggesting that they are being lost. Moreover, intronless genes are less dense in putative ESEs than intron-containing ones. However, this latter difference is likely due to the skewed base composition of intronless sequences, a skew that is in line with the general GC richness of few exon genes. Indeed, after controlling for such biases, we find that both intronless and intron-containing genes are denser in ESEs than expected by chance. Importantly, nucleotide-controlled analysis of evolutionary rates at synonymous sites in ESEs indicates that the ESEs in intronless genes are under purifying selection in both human and mouse. We conclude that on the loss of introns, some but not all, ESE motifs are lost, the remainder having functions beyond a role in splice promotion. These results have implications for the design of intronless transgenes and for understanding the causes of selection on synonymous sites. PMID:26802218

  15. First intron retention in part transcripts of OsEBP-89 gene in tissues of rice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    OsEBP-89 is a transcription factor gene of rice.It contains two introns. Using RT-PCR and Southern hybridization to study OsEBP-89 tissue-specific expression, we found that its first intron (115 bp in length) of its was retained in a fraction of its transcripts of this gene in rice developing seeds. Furthermore, two OsEBP-89 cDNA clones (c89L and c89LH) were screened from a rice cDNA library.Sequence analysis revealed that the first intron was retained in c89L clone, whereas, both the first and second intron sequences were spliced in c89LH. In addition to developing seeds, the first intron unspliced transcripts of OsEBP-89 are detected in leaves and roots of rice, too. However, the ratio of the first intron unspliced to spliced OsEBP-89 transcripts varied in different tissues examined. The potential biological significance of intron retention in OsEBP-89 transcript was discussed.

  16. a Simple Symmetric Algorithm Using a Likeness with Introns Behavior in RNA Sequences

    Science.gov (United States)

    Regoli, Massimo

    2009-02-01

    The RNA-Crypto System (shortly RCS) is a symmetric key algorithm to cipher data. The idea for this new algorithm starts from the observation of nature. In particular from the observation of RNA behavior and some of its properties. The RNA sequences has some sections called Introns. Introns, derived from the term "intragenic regions", are non-coding sections of precursor mRNA (pre-mRNA) or other RNAs, that are removed (spliced out of the RNA) before the mature RNA is formed. Once the introns have been spliced out of a pre-mRNA, the resulting mRNA sequence is ready to be translated into a protein. The corresponding parts of a gene are known as introns as well. The nature and the role of Introns in the pre-mRNA is not clear and it is under ponderous researches by Biologists but, in our case, we will use the presence of Introns in the RNA-Crypto System output as a strong method to add chaotic non coding information and an unnecessary behaviour in the access to the secret key to code the messages. In the RNA-Crypto System algoritnm the introns are sections of the ciphered message with non-coding information as well as in the precursor mRNA.

  17. Pre-Mrna Introns as a Model for Cryptographic Algorithm:. Theory and Experiments

    Science.gov (United States)

    Regoli, Massimo

    2010-01-01

    The RNA-Crypto System (shortly RCS) is a symmetric key algorithm to cipher data. The idea for this new algorithm starts from the observation of nature. In particular from the observation of RNA behavior and some of its properties. In particular the RNA sequences have some sections called Introns. Introns, derived from the term "intragenic regions", are non-coding sections of precursor mRNA (pre-mRNA) or other RNAs, that are removed (spliced out of the RNA) before the mature RNA is formed. Once the introns have been spliced out of a pre-mRNA, the resulting mRNA sequence is ready to be translated into a protein. The corresponding parts of a gene are known as introns as well. The nature and the role of Introns in the pre-mRNA is not clear and it is under ponderous researches by Biologists but, in our case, we will use the presence of Introns in the RNA-Crypto System output as a strong method to add chaotic non coding information and an unnecessary behaviour in the access to the secret key to code the messages. In the RNA-Crypto System algorithm the introns are sections of the ciphered message with non-coding information as well as in the precursor mRNA.

  18. Stable intronic sequence RNAs (sisRNAs): a new layer of gene regulation.

    Science.gov (United States)

    Osman, Ismail; Tay, Mandy Li-Ian; Pek, Jun Wei

    2016-09-01

    Upon splicing, introns are rapidly degraded. Hence, RNAs derived from introns are commonly deemed as junk sequences. However, the discoveries of intronic-derived small nucleolar RNAs (snoRNAs), small Cajal body associated RNAs (scaRNAs) and microRNAs (miRNAs) suggested otherwise. These non-coding RNAs are shown to play various roles in gene regulation. In this review, we highlight another class of intron-derived RNAs known as stable intronic sequence RNAs (sisRNAs). sisRNAs have been observed since the 1980 s; however, we are only beginning to understand their biological significance. Recent studies have shown or suggested that sisRNAs regulate their own host's gene expression, function as molecular sinks or sponges, and regulate protein translation. We propose that sisRNAs function as an additional layer of gene regulation in the cells. PMID:27147469

  19. Assessing the impact of alternative splicing on the diversity and evolution of the proteome in plants

    NARCIS (Netherlands)

    Severing, E.I.

    2011-01-01

    Splicing is one of the key processing steps during the maturation of a gene’s primary transcript into the mRNA molecule used as a template for protein production. Splicing involves the removal of segments called introns and re-joining of the remaining segments called exons. It is by now well e

  20. Protein Trans-Splicing as a Means for Viral Vector-Mediated In Vivo Gene Therapy

    OpenAIRE

    Li, Juan; Sun, Wenchang; Wang, Bing; Xiao, Xiao; Liu, Xiang-Qin

    2008-01-01

    Inteins catalyze protein splicing in a fashion similar to how self-splicing introns catalyze RNA splicing. Split-inteins catalyze precise ligation of two separate polypeptides through trans-splicing in a highly specific manner. Here we report a method of using protein trans-splicing to circumvent the packaging size limit of gene therapy vectors. To demonstrate this method, we chose a large dystrophin gene and an adeno-associated viral (AAV) vector, which has a small packaging size. A highly f...

  1. 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.

  2. Characteristic differences between the promoters of intron-containing and intronless ribosomal protein genes in yeast

    Directory of Open Access Journals (Sweden)

    Vingron Martin

    2008-10-01

    Full Text Available Abstract Background More than two thirds of the highly expressed ribosomal protein (RP genes in Saccharomyces cerevisiae contain introns, which is in sharp contrast to the genome-wide five percent intron-containing genes. It is well established that introns carry regulatory sequences and that the transcription of RP genes is extensively and coordinately regulated. Here we test the hypotheses that introns are innately associated with heavily transcribed genes and that introns of RP genes contribute regulatory TF binding sequences. Moreover, we investigate whether promoter features are significantly different between intron-containing and intronless RP genes. Results We find that directly measured transcription rates tend to be lower for intron-containing compared to intronless RP genes. We do not observe any specifically enriched sequence motifs in the introns of RP genes other than those of the branch point and the two splice sites. Comparing the promoters of intron-containing and intronless RP genes, we detect differences in number and position of Rap1-binding and IFHL motifs. Moreover, the analysis of the length distribution and the folding free energies suggest that, at least in a sub-population of RP genes, the 5' untranslated sequences are optimized for regulatory function. Conclusion Our results argue against the direct involvement of introns in the regulation of transcription of highly expressed genes. Moreover, systematic differences in motif distributions suggest that RP transcription factors may act differently on intron-containing and intronless gene promoters. Thus, our findings contribute to the decoding of the RP promoter architecture and may fuel the discussion on the evolution of introns.

  3. Genome-wide survey of Alternative Splicing in Sorghum Bicolor.

    Science.gov (United States)

    Panahi, Bahman; Abbaszadeh, Bahram; Taghizadeghan, Mehdi; Ebrahimie, Esmaeil

    2014-07-01

    Sorghum bicolor is a member of grass family which is an attractive model plant for genome study due to interesting genome features like low genome size. In this research, we performed comprehensive investigation of Alternative Splicing and ontology aspects of genes those have undergone these events in sorghum bicolor. We used homology based alignments between gene rich transcripts, represented by tentative consensus (TC) transcript sequences, and genomic scaffolds to deduce the structure of genes and identify alternatively spliced transcripts in sorghum. Using homology mapping of assembled expressed sequence tags with genomics data, we identified 2,137 Alternative Splicing events in S. bicolor. Our study showed that complex events and intron retention are the main types of Alternative Splicing events in S. bicolor and highlights the prevalence of splicing site recognition for definition of introns in this plant. Annotations of the alternatively spliced genes revealed that they represent diverse biological process and molecular functions, suggesting a fundamental role for Alternative Splicing in affecting the development and physiology of S. bicolor. PMID:25049459

  4. Did group II intron proliferation in an endosymbiont-bearing archaeon create eukaryotes?

    Directory of Open Access Journals (Sweden)

    Poole Anthony M

    2006-12-01

    Full Text Available Abstract Martin & Koonin recently proposed that the eukaryote nucleus evolved as a quality control mechanism to prevent ribosome readthrough into introns. In their scenario, the bacterial ancestor of mitochondria was resident in an archaeal cell, and group II introns (carried by the fledgling mitochondrion inserted into coding regions in the archaeal host genome. They suggest that if transcription and translation were coupled, and because splicing is expected to have been slower than translation, the effect of insertion would have been ribosome readthrough into introns, resulting in production of aberrant proteins. The emergence of the nuclear compartment would thus have served to separate transcription and splicing from translation, thereby alleviating this problem. In this article, I argue that Martin & Koonin's model is not compatible with current knowledge. The model requires that group II introns would spread aggressively through an archaeal genome. It is well known that selfish elements can spread through an outbreeding sexual population despite a substantial fitness cost to the host. The same is not true for asexual lineages however, where both theory and observation argue that such elements will be under pressure to reduce proliferation, and may be lost completely. The recent introduction of group II introns into archaea by horizontal transfer provides a natural test case with which to evaluate Martin & Koonin's model. The distribution and behaviour of these introns fits prior theoretical expectations, not the scenario of aggressive proliferation advocated by Martin & Koonin. I therefore conclude that the mitochondrial seed hypothesis for the origin of eukaryote introns, on which their model is based, better explains the early expansion of introns in eukaryotes. The mitochondrial seed hypothesis has the capacity to separate the origin of eukaryotes from the origin of introns, leaving open the possibility that the cell that engulfed the

  5. A Splice Region Variant in LDLR Lowers Non-high Density Lipoprotein Cholesterol and Protects against Coronary Artery Disease

    DEFF Research Database (Denmark)

    Gretarsdottir, Solveig; Helgason, Hannes; Helgadottir, Anna;

    2015-01-01

    .7 × 10⁻⁸ and rs17248748-T, OR = 0.92 and Padj = 0.022). The LDLR splice region variant, rs72658867-A, located at position +5 in intron 14 (NM_000527:c.2140+5G>A), causes retention of intron 14 during transcription and is expected to produce a truncated LDL receptor lacking domains essential for function...

  6. Intron-less RNA injected into the nucleus of Xenopus oocytes accesses a regulated translation control pathway.

    OpenAIRE

    Braddock, M; Muckenthaler, M; White, M R; Thorburn, A M; Sommerville, J; Kingsman, A J; Kingsman, S M

    1994-01-01

    The translation of a capped, polyadenylated RNA after injection into the nucleus of Xenopus oocytes occurs only if the RNA contains an intron. A single point mutation in the splice donor site prevents translation. Intron-less RNA is exported efficiently to the cytoplasm and is held, undegraded, in a translationally inert state for several days. Translation can be activated by treating the oocytes with progesterone or by injecting antibodies that bind the FRGY2 class of messenger RNA binding p...

  7. Splicing Programs and Cancer

    OpenAIRE

    Sophie Germann; Lise Gratadou; Martin Dutertre; Didier Auboeuf

    2012-01-01

    Numerous studies report splicing alterations in a multitude of cancers by using gene-by-gene analysis. However, understanding of the role of alternative splicing in cancer is now reaching a new level, thanks to the use of novel technologies allowing the analysis of splicing at a large-scale level. Genome-wide analyses of alternative splicing...

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

    International Nuclear Information System (INIS)

    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. 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.

  10. 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.

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

    Science.gov (United States)

    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 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. PMID:19495418

  12. 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

  13. 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.

  14. C. elegans sequences that control trans-splicing and operon pre-mRNA processing

    OpenAIRE

    Graber, Joel H; Salisbury, Jesse; Hutchins, Lucie N; Blumenthal, Thomas

    2007-01-01

    Many mRNAs in Caenorhabditis elegans are generated through a trans-splicing reaction that adds one of two classes of spliced leader RNA to an independently transcribed pre-mRNA. SL1 leaders are spliced mostly to pre-mRNAs from genes with outrons, intron-like sequences at the 5′-ends of the pre-mRNAs. In contrast, SL2 leaders are nearly exclusively trans-spliced to genes that occur downstream in polycistronic pre-mRNAs produced from operons. Operon pre-mRNA processing requires separation into ...

  15. Fractionation and characterization of a yeast mRNA splicing extract.

    OpenAIRE

    S. C. Cheng; Abelson, J

    1986-01-01

    We have fractionated a yeast whole cell extract that can accurately splice synthetic actin and CYH2 pre-mRNAs. Three fractions, designated I, II, and III, have been separated by use of ammonium sulfate fractionation and chromatography on heparin agarose. Each fraction alone has no splicing activity. Fractions I and II allow the first step of the splicing reaction to proceed, giving rise to the splicing intermediates, free exon 1, and intron-exon 2. Addition of fraction III completes the react...

  16. 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...... better splice site prediction than other available tools. NetAspGene will be very helpful for the study in Aspergillus splice sites and especially in alternative splicing. A webpage for NetAspGene is publicly available at http://www.cbs.dtu.dk/services/NetAspGene....

  17. Sequences necessary for trans-splicing in transiently transfected Brugia malayi

    OpenAIRE

    Liu, Canhui; Oliveira, Ana; Higazi, Tarig B.; Ghedin, Elodie; DePasse, Jay; Thomas R Unnasch

    2007-01-01

    Many genes in parasitic nematodes are both cis- and trans-spliced. Previous studies have demonstrated that a 7nt element encoded in the first intron of the B. malayi 70 kDa heat shock protein (BmHSP70) gene was necessary to permit trans-splicing of transgenic mRNAs in embryos transfected with constructs encoding portions of the BmHSP70 gene. Here we demonstrate that this element (the B. malayi HSP70 trans-splicing motif, or BmHSP70 TSM) is necessary and sufficient to direct trans-splicing of ...

  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. The Agaricus bisporus cox1 gene: the longest mitochondrial gene and the largest reservoir of mitochondrial group i introns.

    Directory of Open Access Journals (Sweden)

    Cyril Férandon

    Full Text Available In eukaryotes, introns are located in nuclear and organelle genes from several kingdoms. Large introns (up to 5 kbp are frequent in mitochondrial genomes of plant and fungi but scarce in Metazoa, even if these organisms are grouped with fungi among the Opisthokonts. Mitochondrial introns are classified in two groups (I and II according to their RNA secondary structure involved in the intron self-splicing mechanism. Most of these mitochondrial group I introns carry a "Homing Endonuclease Gene" (heg encoding a DNA endonuclease acting in transfer and site-specific integration ("homing" and allowing intron spreading and gain after lateral transfer even between species from different kingdoms. Opposed to this gain mechanism, is another which implies that introns, which would have been abundant in the ancestral genes, would mainly evolve by loss. The importance of both mechanisms (loss and gain is matter of debate. Here we report the sequence of the cox1 gene of the button mushroom Agaricus bisporus, the most widely cultivated mushroom in the world. This gene is both the longest mitochondrial gene (29,902 nt and the largest group I intron reservoir reported to date with 18 group I and 1 group II. An exhaustive analysis of the group I introns available in cox1 genes shows that they are mobile genetic elements whose numerous events of loss and gain by lateral transfer combine to explain their wide and patchy distribution extending over several kingdoms. An overview of intron distribution, together with the high frequency of eroded heg, suggests that they are evolving towards loss. In this landscape of eroded and lost intron sequences, the A. bisporus cox1 gene exhibits a peculiar dynamics of intron keeping and catching, leading to the largest collection of mitochondrial group I introns reported to date in a Eukaryote.

  20. Alternative Splice in Alternative Lice.

    Science.gov (United States)

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

    2015-10-01

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

  1. Alternative branch points are selected during splicing of a yeast pre-mRNA in mammalian and yeast extracts.

    OpenAIRE

    Ruskin, B; Pikielny, C W; Rosbash, M; Green, M R

    1986-01-01

    Pre-mRNA splicing in yeast and higher eukaryotes proceeds by similar pathways, in which a probable splicing intermediate and the excised intron are in a lariat configuration. To compare the pre-mRNA splicing mechanisms in yeast and higher eukaryotes, we have analyzed the RNA products resulting from in vitro processing of a yeast intron-containing pre-mRNA in HeLa cell and yeast extracts. In yeast, the RNA branch (2'-5' phosphodiester bond) of the RNA lariat forms at the third adenosine of the...

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

    Science.gov (United States)

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

    2015-04-01

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

  3. Sequence comparison of the rDNA introns from six different species of Tetrahymena

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Engberg, J

    1985-01-01

    We have studied the sequence variation of the rDNA intron among six species of Tetrahymena. From these data, the intron appears to be relatively well conserved in evolution. We have evaluated the sequence variations among the most distant of these species in relation to the secondary structure...... involving compensating base pair changes. Interestingly, one of these is found in a region that is known to be dispensable in the in vitro splicing reaction suggesting differences between the in vivo and in vitro reactions. One of the single nucleotide deletions is found in the so-called "internal guide...

  4. Use of Intron-Disrupted Polyadenylation Sites To Enhance Expression and Safety of Retroviral Vectors

    OpenAIRE

    Ismail, Said I.; Rohll, Jonathan B.; Kingsman, Susan M.; Kingsman, Alan J.; Uden, Mark

    2001-01-01

    Normal mRNA polyadenylation signals are composed of an AAUAAA motif and G/U box spaced 20 to 30 bp apart. If this spacing is increased further, then polyadenylation is disrupted. Previously it has been demonstrated that insertion of an intron will similarly disrupt this signal even though such introns are removed during a nuclear splicing reaction (X. Liu and J. Mertz, Nucleic Acids Res. 21:5256–5263, 1993). This observation has led to the suggestion that polyadenylation site selection is und...

  5. Entropic contributions to the splicing process

    International Nuclear Information System (INIS)

    It has been recently argued that depletion attraction may play an important role in different aspects of cellular organization, ranging from the organization of transcriptional activity in transcription factories to the formation of nuclear bodies. In this paper, we suggest a new application of these ideas in the context of the splicing process, a crucial step of messenger RNA maturation in eukaryotes. We shall show that entropy effects and the resulting depletion attraction may explain the relevance of the aspecific intron length variable in the choice of splice-site recognition modality. On top of that, some qualitative features of the genome architecture of higher eukaryotes can find evolutionary realistic motivation in the light of our model

  6. mRNA-Associated Processes and Their Influence on Exon-Intron Structure in Drosophila melanogaster.

    Science.gov (United States)

    Lepennetier, Gildas; Catania, Francesco

    2016-01-01

    mRNA-associated processes and gene structure in eukaryotes are typically treated as separate research subjects. Here, we bridge this separation and leverage the extensive multidisciplinary work on Drosophila melanogaster to examine the roles that capping, splicing, cleavage/polyadenylation, and telescripting (i.e, the protection of nascent transcripts from premature cleavage/polyadenylation by the splicing factor U1) might play in shaping exon-intron architecture in protein-coding genes. Our findings suggest that the distance between subsequent internal 5' splice sites (5'ss) in Drosophila genes is constrained such that telescripting effects are maximized, in theory, and thus nascent transcripts are less vulnerable to premature termination. Exceptionally weak 5'ss and constraints on intron-exon size at the gene 5' end also indicate that capping might enhance the recruitment of U1 and, in turn, promote telescripting at this location. Finally, a positive correlation between last exon length and last 5'ss strength suggests that optimal donor splice sites in the proximity of the pre-mRNA tail may inhibit the processing of downstream polyadenylation signals more than weak donor splice sites do. These findings corroborate and build upon previous experimental and computational studies on Drosophila genes. They support the possibility, hitherto scantly explored, that mRNA-associated processes impose significant constraints on the evolution of eukaryotic gene structure. PMID:27172210

  7. Regulation of mRNA Levels by Decay-Promoting Introns that Recruit the Exosome Specificity Factor Mmi1

    Directory of Open Access Journals (Sweden)

    Cornelia Kilchert

    2015-12-01

    Full Text Available In eukaryotic cells, inefficient splicing is surprisingly common and leads to the degradation of transcripts with retained introns. How pre-mRNAs are committed to nuclear decay is unknown. Here, we uncover a mechanism by which specific intron-containing transcripts are targeted for nuclear degradation in fission yeast. Sequence elements within these “decay-promoting” introns co-transcriptionally recruit the exosome specificity factor Mmi1, which induces degradation of the unspliced precursor and leads to a reduction in the levels of the spliced mRNA. This mechanism negatively regulates levels of the RNA helicase DDX5/Dbp2 to promote cell survival in response to stress. In contrast, fast removal of decay-promoting introns by co-transcriptional splicing precludes Mmi1 recruitment and relieves negative expression regulation. We propose that decay-promoting introns facilitate the regulation of gene expression. Based on the identification of multiple additional Mmi1 targets, including mRNAs, long non-coding RNAs, and sn/snoRNAs, we suggest a general role in RNA regulation for Mmi1 through transcript degradation.

  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 functional alternative splicing mutation in AIRE gene causes autoimmune polyendocrine syndrome type 1.

    Directory of Open Access Journals (Sweden)

    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.

  10. Distribution of introns in fungal histone genes.

    Directory of Open Access Journals (Sweden)

    Choong-Soo Yun

    Full Text Available Saccharomycotina and Taphrinomycotina lack intron in their histone genes, except for an intron in one of histone H4 genes of Yarrowia lipolytica. On the other hand, Basidiomycota and Perizomycotina have introns in their histone genes. We compared the distributions of 81, 47, 79, and 98 introns in the fungal histone H2A, H2B, H3, and H4 genes, respectively. Based on the multiple alignments of the amino acid sequences of histones, we identified 19, 13, 31, and 22 intron insertion sites in the histone H2A, H2B, H3, and H4 genes, respectively. Surprisingly only one hot spot of introns in the histone H2A gene is shared between Basidiomycota and Perizomycotina, suggesting that most of introns of Basidiomycota and Perizomycotina were acquired independently. Our findings suggest that the common ancestor of Ascomycota and Basidiomycota maybe had a few introns in the histone genes. In the course of fungal evolution, Saccharomycotina and Taphrinomycotina lost the histone introns; Basidiomycota and Perizomycotina acquired other introns independently. In addition, most of the introns have sequence similarity among introns of phylogenetically close species, strongly suggesting that horizontal intron transfer events between phylogenetically distant species have not occurred recently in the fungal histone genes.

  11. Comprehensive splicing graph analysis of alternative splicing patterns in chicken, compared to human and mouse

    Directory of Open Access Journals (Sweden)

    Ranganathan Shoba

    2009-07-01

    Full Text Available Abstract Background Alternative transcript diversity manifests itself as a prime cause of complexity in higher eukaryotes. Recently, transcript diversity studies have suggested that 60–80% of human genes are alternatively spliced. We have used a splicing pattern approach for the bioinformatics analysis of Alternative Splicing (AS in chicken, human and mouse. Exons involved in splicing are subdivided into distinct and variant exons, based on the prevalence of the exons across the transcripts. Four possible permutations of these two different groups of exons were categorised as class I (distinct-variant, class II (distinct-variant, class III (variant-distinct and class IV (variant-variant. This classification quantifies the variation in transcript diversity in the three species. Results In all, 3901 chicken AS genes have been compared with 16,715 human and 16,491 mouse AS genes, with 23% of chicken genes being alternatively spliced, compared to 68% in humans and 57% in mice. To minimize any gene structure bias in the input data, comparative genome analysis has been carried out on the orthologous subset of AS genes for the three species. Gene-level analysis suggested that chicken genes show fewer AS events compared to human and mouse. An event-level analysis showed that the percentage of AS events in chicken is similar to that of human, which implies that a smaller number of chicken genes show greater transcript diversity. Overall, chicken genes were found to have fewer transcripts per gene and shorter introns than human and mouse genes. Conclusion In chicken, the majority of genes generate only two or three isoforms, compared to almost eight in human and six in mouse. We observed that intron definition is expressed strongly when compared to exon definition for chicken genome, based on 3% intron retention in chicken, compared to 2% in human and mouse. Splicing patterns with variant exons account for 33% of AS chicken orthologous genes compared to

  12. Unique splicing pattern of the TCF7L2 gene in human pancreatic islets

    DEFF Research Database (Denmark)

    Osmark, P; Hansson, O; Jonsson, Anna Elisabet;

    2009-01-01

    Intronic variation in the TCF7L2 gene exhibits the strongest association to type 2 diabetes observed to date, but the mechanism whereby this genetic variation translates into altered biological function is largely unknown. A possible explanation is a genotype-dependent difference in the complex...... splicing pattern; however, this has not previously been characterised in pancreatic or insulin target tissues. Here, the detailed TCF7L2 splicing pattern in five human tissues is described and dependence on risk genotype explored....

  13. Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients

    DEFF Research Database (Denmark)

    Petersen, S. M.; Dandanell, M.; Rasmussen, L. J.;

    2013-01-01

    /MSH2 mutations as pathogenic, whereas four MLH1/MSH2/MSH6 mutations are classified as neutral. This study supports the notion that in silico prediction tools and mini-gene assays are important for the classification of intronic variants, and thereby crucial for the genetic counseling of patients and......Background: Germ-line mutations in the DNA mismatch repair genes MLH1, MSH2, and MSH6 predispose to the development of colorectal cancer (Lynch syndrome or hereditary nonpolyposis colorectal cancer). These mutations include disease-causing frame-shift, nonsense, and splicing mutations as well as...... asses the effect on splicing. Results: We describe in silico and in vitro characterization of nine intronic MLH1, MSH2, or MSH6 mutations identified in Danish colorectal cancer patients, of which four mutations are novel. The analysis revealed aberrant splicing of five mutations (MLH1 c. 588 + 5G > A...

  14. Widespread Inhibition of Posttranscriptional Splicing Shapes the Cellular Transcriptome following Heat Shock

    Directory of Open Access Journals (Sweden)

    Reut Shalgi

    2014-06-01

    Full Text Available During heat shock and other proteotoxic stresses, cells regulate multiple steps in gene expression in order to globally repress protein synthesis and selectively upregulate stress response proteins. Splicing of several mRNAs is known to be inhibited during heat stress, often meditated by SRp38, but the extent and specificity of this effect have remained unclear. Here, we examined splicing regulation genome-wide during heat shock in mouse fibroblasts. We observed widespread retention of introns in transcripts from ∼1,700 genes, which were enriched for tRNA synthetase, nuclear pore, and spliceosome functions. Transcripts with retained introns were largely nuclear and untranslated. However, a group of 580+ genes biased for oxidation reduction and protein folding functions continued to be efficiently spliced. Interestingly, these unaffected transcripts are mostly cotranscriptionally spliced under both normal and stress conditions, whereas splicing-inhibited transcripts are mostly spliced posttranscriptionally. Altogether, our data demonstrate widespread repression of splicing in the mammalian heat stress response, disproportionately affecting posttranscriptionally spliced genes.

  15. 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.

  16. A novel splicing mutation alters DSPP transcription and leads to dentinogenesis imperfecta type II.

    Directory of Open Access Journals (Sweden)

    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.

  17. Regulatory mechanisms for 3'-end alternative splicing and polyadenylation of the Glial Fibrillary Acidic Protein, GFAP, transcript

    DEFF Research Database (Denmark)

    Blechingberg, Jenny; Lykke-Andersen, Søren; Jensen, Torben Heick;

    2007-01-01

    molecular mechanisms participating in alternative GFAP expression. Usage of a polyadenylation signal within the alternatively spliced exon 7a is essential to generate the GFAP kappa and GFAP kappa transcripts. The GFAP kappa mRNA is distinct from GFAP epsilon mRNA given that it also includes intron 7a....... Polyadenylation at the exon 7a site is stimulated by the upstream splice site. Moreover, exon 7a splice enhancer motifs supported both exon 7a splicing and polyadenylation. SR proteins increased the usage of the exon 7a polyadenylation signal but not the exon 7a splicing, whereas the polypyrimidine tract binding...... (PTB) protein enhanced both exon 7a polyadenylation and exon 7a splicing. Finally, increasing transcription by the VP16 trans-activator did not affect the frequency of use of the exon 7a polyadenylation signal whereas the exon 7a splicing frequency was decreased. Our data suggest a model with the...

  18. SPA: a probabilistic algorithm for spliced alignment.

    Directory of Open Access Journals (Sweden)

    Erik van Nimwegen

    2006-04-01

    Full Text Available Recent large-scale cDNA sequencing efforts show that elaborate patterns of splice variation are responsible for much of the proteome diversity in higher eukaryotes. To obtain an accurate account of the repertoire of splice variants, and to gain insight into the mechanisms of alternative splicing, it is essential that cDNAs are very accurately mapped to their respective genomes. Currently available algorithms for cDNA-to-genome alignment do not reach the necessary level of accuracy because they use ad hoc scoring models that cannot correctly trade off the likelihoods of various sequencing errors against the probabilities of different gene structures. Here we develop a Bayesian probabilistic approach to cDNA-to-genome alignment. Gene structures are assigned prior probabilities based on the lengths of their introns and exons, and based on the sequences at their splice boundaries. A likelihood model for sequencing errors takes into account the rates at which misincorporation, as well as insertions and deletions of different lengths, occurs during sequencing. The parameters of both the prior and likelihood model can be automatically estimated from a set of cDNAs, thus enabling our method to adapt itself to different organisms and experimental procedures. We implemented our method in a fast cDNA-to-genome alignment program, SPA, and applied it to the FANTOM3 dataset of over 100,000 full-length mouse cDNAs and a dataset of over 20,000 full-length human cDNAs. Comparison with the results of four other mapping programs shows that SPA produces alignments of significantly higher quality. In particular, the quality of the SPA alignments near splice boundaries and SPA's mapping of the 5' and 3' ends of the cDNAs are highly improved, allowing for more accurate identification of transcript starts and ends, and accurate identification of subtle splice variations. Finally, our splice boundary analysis on the human dataset suggests the existence of a novel non

  19. SPA: A Probabilistic Algorithm for Spliced Alignment

    Science.gov (United States)

    van Nimwegen, Erik; Paul, Nicodeme; Sheridan, Robert; Zavolan, Mihaela

    2006-01-01

    Recent large-scale cDNA sequencing efforts show that elaborate patterns of splice variation are responsible for much of the proteome diversity in higher eukaryotes. To obtain an accurate account of the repertoire of splice variants, and to gain insight into the mechanisms of alternative splicing, it is essential that cDNAs are very accurately mapped to their respective genomes. Currently available algorithms for cDNA-to-genome alignment do not reach the necessary level of accuracy because they use ad hoc scoring models that cannot correctly trade off the likelihoods of various sequencing errors against the probabilities of different gene structures. Here we develop a Bayesian probabilistic approach to cDNA-to-genome alignment. Gene structures are assigned prior probabilities based on the lengths of their introns and exons, and based on the sequences at their splice boundaries. A likelihood model for sequencing errors takes into account the rates at which misincorporation, as well as insertions and deletions of different lengths, occurs during sequencing. The parameters of both the prior and likelihood model can be automatically estimated from a set of cDNAs, thus enabling our method to adapt itself to different organisms and experimental procedures. We implemented our method in a fast cDNA-to-genome alignment program, SPA, and applied it to the FANTOM3 dataset of over 100,000 full-length mouse cDNAs and a dataset of over 20,000 full-length human cDNAs. Comparison with the results of four other mapping programs shows that SPA produces alignments of significantly higher quality. In particular, the quality of the SPA alignments near splice boundaries and SPA's mapping of the 5′ and 3′ ends of the cDNAs are highly improved, allowing for more accurate identification of transcript starts and ends, and accurate identification of subtle splice variations. Finally, our splice boundary analysis on the human dataset suggests the existence of a novel non-canonical splice

  20. Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Li, Long; Pintel, David J., E-mail: pinteld@missouri.edu

    2012-04-25

    Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained in unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.

  1. Extensive intron gain in the ancestor of placental mammals

    OpenAIRE

    Kordiš Dušan

    2011-01-01

    Abstract Background Genome-wide studies of intron dynamics in mammalian orthologous genes have found convincing evidence for loss of introns but very little for intron turnover. Similarly, large-scale analysis of intron dynamics in a few vertebrate genomes has identified only intron losses and no gains, indicating that intron gain is an extremely rare event in vertebrate evolution. These studies suggest that the intron-rich genomes of vertebrates do not allow intron gain. The aim of this stud...

  2. Towards understanding pre-mRNA splicing mechanisms and the role of SR proteins.

    Science.gov (United States)

    Sahebi, Mahbod; Hanafi, Mohamed M; van Wijnen, Andre J; Azizi, Parisa; Abiri, Rambod; Ashkani, Sadegh; Taheri, Sima

    2016-08-10

    Alternative pre-mRNA splicing provides a source of vast protein diversity by removing non-coding sequences (introns) and accurately linking different exonic regions in the correct reading frame. The regulation of alternative splicing is essential for various cellular functions in both pathological and physiological conditions. In eukaryotic cells, this process is commonly used to increase proteomic diversity and to control gene expression either co- or post-transcriptionally. Alternative splicing occurs within a megadalton-sized, multi-component machine consisting of RNA and proteins; during the splicing process, this complex undergoes dynamic changes via RNA-RNA, protein-protein and RNA-protein interactions. Co-transcriptional splicing functionally integrates the transcriptional machinery, thereby enabling the two processes to influence one another, whereas post-transcriptional splicing facilitates the coupling of RNA splicing with post-splicing events. This review addresses the structural aspects of spliceosomes and the mechanistic implications of their stepwise assembly on the regulation of pre-mRNA splicing. Moreover, the role of phosphorylation-based, signal-induced changes in the regulation of the splicing process is demonstrated. PMID:27154819

  3. Complex Alternative Splicing

    OpenAIRE

    Park, Jung Woo; Graveley, Brenton R.

    2007-01-01

    Alternative splicing is a powerful means of controlling gene expression and increasing protein diversity. Most genes express a limited number of mRNA isoforms, but there are several examples of genes that use alternative splicing to generate hundreds, thousands, and even tens of thousands of isoforms. Collectively such genes are considered to undergo complex alternative splicing. The best example is the Drosophila Down syndrome cell adhesion molecule (Dscam) gene, which can generate 38,016 is...

  4. Where splicing joins chromatin

    OpenAIRE

    Hnilicová, Jarmila; Staněk, David

    2011-01-01

    There are numerous data suggesting that two key steps in gene expression—transcription and splicing influence each other closely. For a long time it was known that chromatin modifications regulate transcription, but only recently it was shown that chromatin and histone modifications play a significant role in pre-mRNA splicing. Here we summarize interactions between splicing machinery and chromatin and discuss their potential functional significance. We focus mainly on histone acetylation and...

  5. Kinetin improves IKBKAP mRNA splicing in patients with familial dysautonomia

    OpenAIRE

    Axelrod, Felicia B.; Liebes, Leonard; Gold-von Simson, Gabrielle; Mendoza, Sandra; Mull, James; Leyne, Maire; Norcliffe-Kaufmann, Lucy; Kaufmann, Horacio; Slaugenhaupt, Susan A.

    2011-01-01

    Familial dysautonomia (FD) is caused by an intronic splice mutation in the IKBKAP gene that leads to partial skipping of exon 20 and tissue-specific reduction in I-κ-B kinase complex associated protein/ elongation protein 1 (IKAP/ELP-1) expression. Kinetin (6-furfurylaminopurine) has been shown to improve splicing and increase wild-type IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine if oral kinetin treatment could alter mRNA splicing in FD subjects and was...

  6. Tafazzin splice variants and mutations in Barth syndrome.

    Science.gov (United States)

    Kirwin, Susan M; Manolakos, Athena; Barnett, Sarah Swain; Gonzalez, Iris L

    2014-01-01

    Barth syndrome is caused by mutations in the TAZ (tafazzin) gene on human chromosome Xq28. The human tafazzin gene produces four major mRNA splice variants; two of which have been shown to be functional (TAZ lacking exon 5 and full-length) in complementation studies with yeast and Drosophila. This study characterizes the multiple alternative splice variants of TAZ mRNA and their proportions in blood samples from a cohort of individuals with Barth syndrome (BTHS). Because it has been reported that collection and processing methods can affect the expression of various genes, we tested and chose a stabilizing medium for collecting, shipping and processing of the blood samples of these individuals. In both healthy controls and in BTHS individuals, we found a greater variety of alternatively spliced forms than previously described, with a sizeable proportion of minor splice variants besides the four dominant isoforms. Individuals with certain exonic and intronic splice mutations produce additional mutant mRNAs that could be translated into two or more proteins with different amino acid substitutions in a single individual. A fraction of the minor splice variants is predicted to be non-productive. PMID:24342716

  7. 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 in...... patterns of splicing. The functionality of the vast majority of cassette exons is underscored by various other features. We suggest that differences in conservation between lineages reflect differences in levels of functionality and further suggest that these differences are due to differences in intron...... length and the strength of consensus boundaries across lineages. Finally, we demonstrate an inverse relationship between AS and gene duplication, suggesting that the latter may be primarily responsible for the emergence of new functional transcripts in nematodes. Udgivelsesdato: 2008-Feb...

  8. Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene

    Science.gov (United States)

    Känsäkoski, Johanna; Jääskeläinen, Jarmo; Jääskeläinen, Tiina; Tommiska, Johanna; Saarinen, Lilli; Lehtonen, Rainer; Hautaniemi, Sampsa; Frilander, Mikko J.; Palvimo, Jorma J.; Toppari, Jorma; Raivio, Taneli

    2016-01-01

    Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5′ splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation. PMID:27609317

  9. Recurrent insertion of 5'-terminal nucleotides and loss of the branchpoint motif in lineages of group II introns inserted in mitochondrial preribosomal RNAs.

    Science.gov (United States)

    Li, Cheng-Fang; Costa, Maria; Bassi, Gurminder; Lai, Yiu-Kay; Michel, François

    2011-07-01

    A survey of sequence databases revealed 10 instances of subgroup IIB1 mitochondrial ribosomal introns with 1 to 33 additional nucleotides inserted between the 5' exon and the consensus sequence at the intron 5' end. These 10 introns depart further from the IIB1 consensus in their predicted domain VI structure: In contrast to its basal helix and distal GNRA terminal loop, the middle part of domain VI is highly variable and lacks the bulging A that serves as the branchpoint in lariat formation. In vitro experiments using two closely related IIB1 members inserted at the same ribosomal RNA site in the basidiomycete fungi Grifola frondosa and Pycnoporellus fulgens revealed that both ribozymes are capable of efficient self-splicing. However, whereas the Grifola intron was excised predominantly as a lariat, the Pycnoporellus intron, which possesses six additional nucleotides at the 5' end, yielded only linear products, consistent with its predicted domain VI structure. Strikingly, all of the introns with 5' terminal insertions lack the EBS2 exon-binding site. Moreover, several of them are part of the small subset of group II introns that encode potentially functional homing endonucleases of the LAGLIDADG family rather than reverse transcriptases. Such coincidences suggest causal relationships between the shift to DNA-based mobility, the loss of one of the two ribozyme sites for binding the 5' exon, and the exclusive use of hydrolysis to initiate splicing. PMID:21613530

  10. 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...

  11. Intron 4 containing novel GABAB1 isoforms impair GABAB receptor function.

    Directory of Open Access Journals (Sweden)

    Changhoon Lee

    Full Text Available BACKGROUND: Gamma-aminobutyric acid type B (GABAB receptors decrease neural activity through G protein signaling. There are two subunits, GABAB1 and GABAB2. Alternative splicing provides GABAB1 with structural and functional diversity. cDNA microarrays showed strong signals from human brain RNA using GABAB1 intron 4 region probes. Therefore, we predicted the existence of novel splice variants. METHODOLOGY/PRINCIPAL FINDINGS: Based on the probe sequence analysis, we proposed two possible splice variants, GABAB1j and GABAB1k. The existence of human GABAB1j was verified by quantitative real-time PCR, and mouse GABAB1j was found from a microarray probe set based on human GABAB1j sequence. GABAB1j open reading frames (ORF and expression patterns are not conserved across species, and they do not have any important functional domains except sushi domains. Thus, we focused on another possible splice variant, GABAB1k. After obtaining PCR evidence for GABAB1k existence from human, mouse, and rat, it was cloned from human and mouse by PCR along with three additional isoforms, GABAB1l, GABAB1m, and GABAB1n. Their expression levels by quantitative real-time PCR are relatively low in brain although they may be expressed in specific cell types. GABAB1l and GABAB1m inhibit GABAB receptor-induced G protein-activated inwardly rectifying K(+ channel (GIRK currents at Xenopus oocyte two-electrode voltage clamp system. CONCLUSIONS/SIGNIFICANCE: This study supports previous suggestions that intron 4 of GABAB1 gene is a frequent splicing spot across species. Like GABAB1e, GABAB1l and GABAB1m do not have transmembrane domains but have a dimerization motif. So, they also could be secreted and bind GABAB2 dominantly instead of GABAB1a. However, only GABAB1l and GABAB1m are N- and C-terminal truncated splicing variants and impair receptor function. This suggests that the intron 4 containing N-terminal truncation is necessary for the inhibitory action of the new splice

  12. Phylogenetic evidence for the acquisition of ribosomal RNA introns subsequent to the divergence of some of the major Tetrahymena groups

    DEFF Research Database (Denmark)

    Sogin, M L; Ingold, A; Karlok, M;

    1986-01-01

    Previous work has demonstrated the presence of a self-splicing intron in the large subunit ribosomal RNA coding region in some strains of the ciliate protozoan Tetrahymena. Sequence comparisons of the intron regions from six Tetrahymena species showed these to fall into three homology groups. In an...... attempt to evaluate the evolutionary origins of the intervening sequences, we have now determined complete small subunit ribosomal RNA gene sequences from 13 species of Tetrahymena and the absolute number of nucleotide differences between the sequences was used to construct a phylogenetic tree. This...

  13. The proper splicing of RNAi factors is critical for pericentric heterochromatin assembly in fission yeast.

    Directory of Open Access Journals (Sweden)

    Scott P Kallgren

    Full Text Available Heterochromatin preferentially assembles at repetitive DNA elements, playing roles in transcriptional silencing, recombination suppression, and chromosome segregation. The RNAi machinery is required for heterochromatin assembly in a diverse range of organisms. In fission yeast, RNA splicing factors are also required for pericentric heterochromatin assembly, and a prevailing model is that splicing factors provide a platform for siRNA generation independently of their splicing activity. Here, by screening the fission yeast deletion library, we discovered four novel splicing factors that are required for pericentric heterochromatin assembly. Sequencing total cellular RNAs from the strongest of these mutants, cwf14Δ, showed intron retention in mRNAs of several RNAi factors. Moreover, introducing cDNA versions of RNAi factors significantly restored pericentric heterochromatin in splicing mutants. We also found that mutations of splicing factors resulted in defective telomeric heterochromatin assembly and mis-splicing the mRNA of shelterin component Tpz1, and that replacement of tpz1+ with its cDNA partially rescued heterochromatin defects at telomeres in splicing mutants. Thus, proper splicing of RNAi and shelterin factors contributes to heterochromatin assembly at pericentric regions and telomeres.

  14. Trans-Splicing Improvement by the Combined Application of Antisense Strategies

    Directory of Open Access Journals (Sweden)

    Ulrich Koller

    2015-01-01

    Full Text Available Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs, presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB. We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG, lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

  15. 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.

  16. 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

    experimentally observed in A.thaliana transformants. Predictions for alternatively spliced genes are also presented, together with examples of genes from other dicots, monocots and algae. The method has been made available through electronic mail (NetPlantGene@cbs.dtu.dk), or the WWW at http://www.cbs.dtu.dk/NetPlantGene.html......Artificial neural networks have been combined with a rule based system to predict intron splice sites in the dicot plant Arabidopsis thaliana. A two step prediction scheme, where a global prediction of the coding potential regulates a cutoff level for a local predicition of splice sites, is refined...... by rules based on splice site confidence values, prediction scores, coding context and distances between potential splice sites. In this approach, the prediction of splice sites mutually affect each other in a non-local manner. The combined approach drastically reduces the large amount of false...

  17. SpliceDisease database: linking RNA splicing and disease

    OpenAIRE

    WANG, Juan; Jie ZHANG; Li, Kaibo; Zhao, Wei; Cui, Qinghua

    2011-01-01

    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 associa...

  18. Novel Introner-Like Elements in fungi Are Involved in Parallel Gains of Spliceosomal Introns

    OpenAIRE

    Collemare, J.; Beenen, H.G.; Crous, P.W.; Wit, de, Joost; Burgt, van der, G.J.H.M.

    2015-01-01

    Spliceosomal introns are key components of the eukaryotic gene structure. Although they contributed to the emergence of eukaryotes, their origin remains elusive. In fungi, they might originate from the multiplication of invasive introns named Introner-Like Elements (ILEs). However, so far ILEs have been observed in six fungal species only, including Fulvia fulva and Dothistroma septosporum (Dothideomycetes), arguing against ILE insertion as a general mechanism for intron gain. Here, we identi...

  19. Intron Evolution: Testing Hypotheses of Intron Evolution Using the Phylogenomics of Tetraspanins

    OpenAIRE

    Antonio Garcia-España; Roso Mares; Tung-Tien Sun; Rob Desalle

    2009-01-01

    BACKGROUND: Although large scale informatics studies on introns can be useful in making broad inferences concerning patterns of intron gain and loss, more specific questions about intron evolution at a finer scale can be addressed using a gene family where structure and function are well known. Genome wide surveys of tetraspanins from a broad array of organisms with fully sequenced genomes are an excellent means to understand specifics of intron evolution. Our approach incorporated several ne...

  20. Splicing regulators: targets and drugs

    OpenAIRE

    Yeo, Gene Wei-Ming

    2005-01-01

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

  1. RNA binding specificity of hnRNP proteins: a subset bind to the 3' end of introns.

    OpenAIRE

    Swanson, M S; Dreyfuss, G

    1988-01-01

    The binding of hnRNP proteins to pre-mRNAs in nuclear extracts, and as isolated proteins, was studied by using monoclonal antibody immunopurification of hnRNP proteins bound to RNase T1-generated fragments. Several major hnRNP proteins, A1, C and D, bind specifically to the 3' end of introns within a region containing the conserved polypyrimidine stretch between the branch site and the 3' splice site. Mutations which alter the conserved 3' splice site dinucleotide AG strongly impair or abolis...

  2. 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. PMID:18404972

  3. Progress on research of the alternative splicing of human cytochrome P450 pre-mRNA%人细胞色素P450前mRNA的可变剪接研究进展

    Institute of Scientific and Technical Information of China (English)

    诸葛坚; 余应年

    2005-01-01

    Human genes typically contain multiple introns, and in many cases the exons can be joined more than one way to generate multiple rnRNAs, encoding distinct protein isoforms. This process is called alternative splicing. The article summarized the human cytochrome P450 pre-mRNA alternative splicing and their regulatory mechanism and impacts on biological functions.

  4. 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.

  5. 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

  6. 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.

  7. Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease.

    Directory of Open Access Journals (Sweden)

    Tuhin Kumar Guha

    Full Text Available In Chaetomium thermophilum (DSM 1495 within the mitochondrial DNA (mtDNA small ribosomal subunit (rns gene a group IIA1 intron interrupts an open reading frame (ORF encoded within a group I intron (mS1247. This arrangement offers the opportunity to examine if the nested group II intron could be utilized as a regulatory element for the expression of the homing endonuclease (HEase. Constructs were generated where the codon-optimized ORF was interrupted with either the native group IIA1 intron or a group IIB type intron. This study showed that the expression of the HEase (in vivo in Escherichia coli can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. Exogenous magnesium chloride (MgCl2 stimulated the expression of a functional HEase but the addition of cobalt chloride (CoCl2 to growth media antagonized the expression of HEase activity. Ultimately the ability to attenuate HEase activity might be useful in precision genome engineering, minimizing off target activities, or where pathways have to be altered during a specific growth phase.

  8. Suppression of the Arboviruses Dengue and Chikungunya Using a Dual-Acting Group-I Intron Coupled with Conditional Expression of the Bax C-Terminal Domain.

    Directory of Open Access Journals (Sweden)

    James R Carter

    Full Text Available In portions of South Asia, vectors and patients co-infected with dengue (DENV and chikungunya (CHIKV are on the rise, with the potential for this occurrence in other regions of the world, for example the United States. Therefore, we engineered an antiviral approach that suppresses the replication of both arboviruses in mosquito cells using a single antiviral group I intron. We devised unique configurations of internal, external, and guide sequences that permit homologous recognition and splicing with conserved target sequences in the genomes of both viruses using a single trans-splicing Group I intron, and examined their effectiveness to suppress infections of DENV and CHIKV in mosquito cells when coupled with a proapoptotic 3' exon, ΔN Bax. RT-PCR demonstrated the utility of these introns in trans-splicing the ΔN Bax sequence downstream of either the DENV or CHIKV target site in transformed Aedes albopictus C6/36 cells, independent of the order in which the virus specific targeting sequences were inserted into the construct. This trans-splicing reaction forms DENV or CHIKV ΔN Bax RNA fusions that led to apoptotic cell death as evidenced by annexin V staining, caspase, and DNA fragmentation assays. TCID50-IFA analyses demonstrate effective suppression of DENV and CHIKV infections by our anti-arbovirus group I intron approach. This represents the first report of a dual-acting Group I intron, and demonstrates that we can target DENV and CHIKV RNAs in a sequence specific manner with a single, uniquely configured CHIKV/DENV dual targeting group I intron, leading to replication suppression of both arboviruses, and thus providing a promising single antiviral for the transgenic suppression of multiple arboviruses.

  9. Splicing-Mediated Autoregulation Modulates Rpl22p Expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Gabunilas, Jason; Chanfreau, Guillaume

    2016-04-01

    In Saccharomyces cerevisiae, splicing is critical for expression of ribosomal protein genes (RPGs), which are among the most highly expressed genes and are tightly regulated according to growth and environmental conditions. However, knowledge of the precise mechanisms by which RPG pre-mRNA splicing is regulated on a gene-by-gene basis is lacking. Here we show that Rpl22p has an extraribosomal role in the inhibition of splicing of the RPL22B pre-mRNA transcript. A stem loop secondary structure within the intron is necessary for pre-mRNA binding by Rpl22p in vivo and splicing inhibition in vivo and in vitro and can rescue splicing inhibition in vitro when added in trans to splicing reactions. Splicing inhibition by Rpl22p may be partly attributed to the reduction of co-transcriptional U1 snRNP recruitment to the pre-mRNA at the RPL22B locus. We further demonstrate that the inhibition of RPL22B pre-mRNA splicing contributes to the down-regulation of mature transcript during specific stress conditions, and provide evidence hinting at a regulatory role for this mechanism in conditions of suppressed ribosome biogenesis. These results demonstrate an autoregulatory mechanism that fine-tunes the expression of the Rpl22 protein and by extension Rpl22p paralog composition according to the cellular demands for ribosome biogenesis. PMID:27097027

  10. Histone H3K36 methylation regulates pre-mRNA splicing in Saccharomyces cerevisiae.

    Science.gov (United States)

    Sorenson, Matthew R; Jha, Deepak K; Ucles, Stefanie A; Flood, Danielle M; Strahl, Brian D; Stevens, Scott W; Kress, Tracy L

    2016-04-01

    Co-transcriptional splicing takes place in the context of a highly dynamic chromatin architecture, yet the role of chromatin restructuring in coordinating transcription with RNA splicing has not been fully resolved. To further define the contribution of histone modifications to pre-mRNA splicing in Saccharomyces cerevisiae, we probed a library of histone point mutants using a reporter to monitor pre-mRNA splicing. We found that mutation of H3 lysine 36 (H3K36) - a residue methylated by Set2 during transcription elongation - exhibited phenotypes similar to those of pre-mRNA splicing mutants. We identified genetic interactions between genes encoding RNA splicing factors and genes encoding the H3K36 methyltransferase Set2 and the demethylase Jhd1 as well as point mutations of H3K36 that block methylation. Consistent with the genetic interactions, deletion of SET2, mutations modifying the catalytic activity of Set2 or H3K36 point mutations significantly altered expression of our reporter and reduced splicing of endogenous introns. These effects were dependent on the association of Set2 with RNA polymerase II and H3K36 dimethylation. Additionally, we found that deletion of SET2 reduces the association of the U2 and U5 snRNPs with chromatin. Thus, our study provides the first evidence that H3K36 methylation plays a role in co-transcriptional RNA splicing in yeast. PMID:26821844

  11. Analysis of a splice array experiment elucidates roles of chromatin elongation factor Spt4-5 in splicing.

    Directory of Open Access Journals (Sweden)

    Yuanyuan Xiao

    2005-09-01

    Full Text Available Splicing is an important process for regulation of gene expression in eukaryotes, and it has important functional links to other steps of gene expression. Two examples of these linkages include Ceg1, a component of the mRNA capping enzyme, and the chromatin elongation factors Spt4-5, both of which have recently been shown to play a role in the normal splicing of several genes in the yeast Saccharomyces cerevisiae. Using a genomic approach to characterize the roles of Spt4-5 in splicing, we used splicing-sensitive DNA microarrays to identify specific sets of genes that are mis-spliced in ceg1, spt4, and spt5 mutants. In the context of a complex, nested, experimental design featuring 22 dye-swap array hybridizations, comprising both biological and technical replicates, we applied five appropriate statistical models for assessing differential expression between wild-type and the mutants. To refine selection of differential expression genes, we then used a robust model-synthesizing approach, Differential Expression via Distance Synthesis, to integrate all five models. The resultant list of differentially expressed genes was then further analyzed with regard to select attributes: we found that highly transcribed genes with long introns were most sensitive to spt mutations. QPCR confirmation of differential expression was established for the limited number of genes evaluated. In this paper, we showcase splicing array technology, as well as powerful, yet general, statistical methodology for assessing differential expression, in the context of a real, complex experimental design. Our results suggest that the Spt4-Spt5 complex may help coordinate splicing with transcription under conditions that present kinetic challenges to spliceosome assembly or function.

  12. 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. PMID:26016411

  13. Use of intron-disrupted polyadenylation sites to enhance expression and safety of retroviral vectors.

    Science.gov (United States)

    Ismail, S I; Rohll, J B; Kingsman, S M; Kingsman, A J; Uden, M

    2001-01-01

    Normal mRNA polyadenylation signals are composed of an AAUAAA motif and G/U box spaced 20 to 30 bp apart. If this spacing is increased further, then polyadenylation is disrupted. Previously it has been demonstrated that insertion of an intron will similarly disrupt this signal even though such introns are removed during a nuclear splicing reaction (X. Liu and J. Mertz, Nucleic Acids Res. 21:5256-5263, 1993). This observation has led to the suggestion that polyadenylation site selection is undertaken prior to intron excision. We now present results that both support and extend these observations and in doing so create a novel class of retroviral expression vector with improved qualities. We found that when an intron-disrupted polyadenylation signal is inserted within a retroviral expression vector, such a signal, although reformed in the producer cell, remains benign until transduction, where it is then preferentially used. Thus, we demonstrate that upon transduction these vectors now produce a majority of shortened subgenomic species and as a consequence have a reduced tendency for subsequent mobilization from transduced cells. In addition, we demonstrate that the use of this internal signal leads to enhanced expression from such vectors and that this is achieved without any loss in titer. Therefore, split polyadenylation signals confer enhanced performance and improved safety upon retroviral expression vectors into which they are inserted. Such split signals may prove useful for the future optimization of retroviral vectors in gene therapy. PMID:11119589

  14. SNW1 enables sister chromatid cohesion by mediating the splicing of sororin and APC2 pre-mRNAs

    OpenAIRE

    van der Lelij, Petra; Stocsits, Roman R; Ladurner, Rene; Petzold, Georg; Kreidl, Emanuel; Koch, Birgit; Schmitz, Julia; Neumann, Beate; Ellenberg, Jan; Peters, Jan-Michael

    2014-01-01

    Although splicing is essential for the expression of most eukaryotic genes, inactivation of splicing factors causes specific defects in mitosis. The molecular cause of this defect is unknown. Here, we show that the spliceosome subunits SNW1 and PRPF8 are essential for sister chromatid cohesion in human cells. A transcriptome-wide analysis revealed that SNW1 or PRPF8 depletion affects the splicing of specific introns in a subset of pre-mRNAs, including pre-mRNAs encoding the cohesion protein s...

  15. Pre-mRNA trans-splicing: from kinetoplastids to mammals, an easy language for life diversity

    Directory of Open Access Journals (Sweden)

    Mario Gustavo Mayer

    2005-08-01

    Full Text Available Since the discovery that genes are split into intron and exons, the studies of the mechanisms involved in splicing pointed to presence of consensus signals in an attempt to generalize the process for all living cells. However, as discussed in the present review, splicing is a theme full of variations. The trans-splicing of pre-mRNAs, the joining of exons from distinct transcripts, is one of these variations with broad distribution in the phylogenetic tree. The biological meaning of this phenomenon is discussed encompassing reactions resembling a possible noise to mechanisms of gene expression regulation. All of them however, can contribute to the generation of life diversity.

  16. Survey on Nucleotide Encoding Techniques and SVM Kernel Design for Human Splice Site Prediction

    Directory of Open Access Journals (Sweden)

    A.T.M. Golam Bari

    2012-12-01

    Full Text Available Splice site prediction in DNA sequence is a basic search problem for finding exon/intron and intron/exon boundaries. Removing introns and then joining the exons together forms the mRNA sequence. These sequences are the input of the translation process. It is a necessary step in the central dogma of molecular biology. The main task of splice site prediction is to find out the exact GT and AG ended sequences. Then it identifies the true and false GT and AG ended sequences among those candidate sequences. In this paper, we survey research works on splice site prediction based on support vector machine (SVM. The basic difference between these research works is nucleotide encoding technique and SVM kernel selection. Some methods encode the DNA sequence in a sparse way whereas others encode in a probabilistic manner. The encoded sequences serve as input of SVM. The task of SVM is to classify them using its learning model. The accuracy of classification largely depends on the proper kernel selection for sequence data as well as a selection of kernel parameter. We observe each encoding technique and classify them according to their similarity. Then we discuss about kernel and their parameter selection. Our survey paper provides a basic understanding of encoding approaches and proper kernel selection of SVM for splice site prediction.

  17. Regulation of alternative splicing of Bcl-x by IL-6, GM-CSF and TPA

    Institute of Scientific and Technical Information of China (English)

    Chang You LI; Jia You CHU; Jian Kun YU; Xiao Qin HUANG; Xiao Juan LIU; Li SHI; Yan Chun CHE; Jiu Yong XIE

    2004-01-01

    The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of Bcl-x pre-mRNA splicing by extracellular factors and their distinctrequirements for pre-mRNA elements. In K562 leukemia cells, treatment with interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF) reduced the proportion of the Bcl-xL variant mRNA while treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) had no effect. In U251 glioma cells, however, TPA efficientlyincreased the Bcl-xL level. These regulations were also seen for a transfected splicing reporter mini-gene. Further analyses of deletion mutants indicate that nucleotides 1-176 of the downstream intron are required for the IL-6 effect, whereas additional nucleotides 177-284 are essential for the GM-CSF effect. As for the TPA effect, only nucleotides 1-76 are required in the downstream intron. Thus, IL-6, GM-CSF and TPA differentially regulate Bcl-x splicing and require specific intronic pre-mRNA sequences for their respective effects.

  18. Single-molecule RNA observation in vivo reveals dynamics of co-transcriptional splicing

    Science.gov (United States)

    Ferguson, M. L.; Coulon, A.; de Turris, V.; Palangat, M.; Chow, C. C.; Singer, R. H.; Larson, D. R.

    2013-03-01

    The synthesis of pre-mRNA and the splicing of that pre-mRNA to form completed transcripts requires coordination between two large multi-subunit complexes (the transcription elongation complex and the spliceosome). How this coordination occurs in vivo is unknown. Here we report the first experimental observation of transcription and splicing occurring at the same gene in living cells. By utilizing the PP7/MS2 fluorescent RNA reporter system, we can directly observe two distinct regions of the nascent RNA, allowing us to measure the rise and fall time of the intron and exon of a reporter gene stably integrated into a human cell line. The reporter gene consists of a beta globin gene where we have inserted a 24 RNA hairpin cassette into the intron/exon. Upon synthesis, the RNA hairpins are tightly bound by fluorescently-labeled PP7/MS2 bacteriophage coat proteins. After gene induction, a single locus of active transcription in the nucleus shows fluorescence intensity changes characteristic of the synthesis and excision of the intron/exon. Using fluctuation analysis, we determine the elongation rate to be 1.5 kb/min. From the temporal cross correlation function, we determine that splicing of this gene must be co-transcriptional with a splicing time of ~100 seconds before termination and a ~200 second pause at termination. We propose that dual-color RNA imaging may be extended to investigate other mechanisms of transcription, gene regulation, and RNA processing.

  19. Mutations affecting excision of the intron from a eukaryotic dimeric tRNA precursor.

    OpenAIRE

    Willis, I; Hottinger, H; Pearson, D.; Chisholm, V; Leupold, U; Söll, D

    1984-01-01

    The nucleotide sequences of a Schizosaccharomyces pombe opal suppressor serine tRNA gene (sup9-e) and of 12 in vivo-generated mutant genes, which have lost the ability to suppress UGA mutations, have been determined. Analysis of the expression of these genes in Saccharomyces cerevisiae in vitro and in vivo systems has revealed defects in tRNA gene transcription and precursor tRNA processing. Single base changes in the D-loop, the intron and the extra arm affect the efficiency of splicing of t...

  20. Regulation of expression of two LY-6 family genes by intron retention and transcription induced chimerism

    Directory of Open Access Journals (Sweden)

    Mallya Meera

    2008-09-01

    Full Text Available Abstract Background Regulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues. Results The MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD. This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC. This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKβ-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively. Conclusion In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKβ or G6F undergo differential splicing only in the context of the chimera (CSNKβ-LY6G5B or G6F-LY6G6C and not on their own.

  1. Diverse forms of RPS9 splicing are part of an evolving autoregulatory circuit.

    Science.gov (United States)

    Plocik, Alex M; Guthrie, Christine

    2012-01-01

    Ribosomal proteins are essential to life. While the functions of ribosomal protein-encoding genes (RPGs) are highly conserved, the evolution of their regulatory mechanisms is remarkably dynamic. In Saccharomyces cerevisiae, RPGs are unusual in that they are commonly present as two highly similar gene copies and in that they are over-represented among intron-containing genes. To investigate the role of introns in the regulation of RPG expression, we constructed 16 S. cerevisiae strains with precise deletions of RPG introns. We found that several yeast introns function to repress rather than to increase steady-state mRNA levels. Among these, the RPS9A and RPS9B introns were required for cross-regulation of the two paralogous gene copies, which is consistent with the duplication of an autoregulatory circuit. To test for similar intron function in animals, we performed an experimental test and comparative analyses for autoregulation among distantly related animal RPS9 orthologs. Overexpression of an exogenous RpS9 copy in Drosophila melanogaster S2 cells induced alternative splicing and degradation of the endogenous copy by nonsense-mediated decay (NMD). Also, analysis of expressed sequence tag data from distantly related animals, including Homo sapiens and Ciona intestinalis, revealed diverse alternatively-spliced RPS9 isoforms predicted to elicit NMD. We propose that multiple forms of splicing regulation among RPS9 orthologs from various eukaryotes operate analogously to translational repression of the alpha operon by S4, the distant prokaryotic ortholog. Thus, RPS9 orthologs appear to have independently evolved variations on a fundamental autoregulatory circuit. PMID:22479208

  2. Diverse forms of RPS9 splicing are part of an evolving autoregulatory circuit.

    Directory of Open Access Journals (Sweden)

    Alex M Plocik

    Full Text Available Ribosomal proteins are essential to life. While the functions of ribosomal protein-encoding genes (RPGs are highly conserved, the evolution of their regulatory mechanisms is remarkably dynamic. In Saccharomyces cerevisiae, RPGs are unusual in that they are commonly present as two highly similar gene copies and in that they are over-represented among intron-containing genes. To investigate the role of introns in the regulation of RPG expression, we constructed 16 S. cerevisiae strains with precise deletions of RPG introns. We found that several yeast introns function to repress rather than to increase steady-state mRNA levels. Among these, the RPS9A and RPS9B introns were required for cross-regulation of the two paralogous gene copies, which is consistent with the duplication of an autoregulatory circuit. To test for similar intron function in animals, we performed an experimental test and comparative analyses for autoregulation among distantly related animal RPS9 orthologs. Overexpression of an exogenous RpS9 copy in Drosophila melanogaster S2 cells induced alternative splicing and degradation of the endogenous copy by nonsense-mediated decay (NMD. Also, analysis of expressed sequence tag data from distantly related animals, including Homo sapiens and Ciona intestinalis, revealed diverse alternatively-spliced RPS9 isoforms predicted to elicit NMD. We propose that multiple forms of splicing regulation among RPS9 orthologs from various eukaryotes operate analogously to translational repression of the alpha operon by S4, the distant prokaryotic ortholog. Thus, RPS9 orthologs appear to have independently evolved variations on a fundamental autoregulatory circuit.

  3. Intron evolution: testing hypotheses of intron evolution using the phylogenomics of tetraspanins.

    Directory of Open Access Journals (Sweden)

    Antonio Garcia-España

    Full Text Available BACKGROUND: Although large scale informatics studies on introns can be useful in making broad inferences concerning patterns of intron gain and loss, more specific questions about intron evolution at a finer scale can be addressed using a gene family where structure and function are well known. Genome wide surveys of tetraspanins from a broad array of organisms with fully sequenced genomes are an excellent means to understand specifics of intron evolution. Our approach incorporated several new fully sequenced genomes that cover the major lineages of the animal kingdom as well as plants, protists and fungi. The analysis of exon/intron gene structure in such an evolutionary broad set of genomes allowed us to identify ancestral intron structure in tetraspanins throughout the eukaryotic tree of life. METHODOLOGY/PRINCIPAL FINDINGS: We performed a phylogenomic analysis of the intron/exon structure of the tetraspanin protein family. In addition, to the already characterized tetraspanin introns numbered 1 through 6 found in animals, three additional ancient, phase 0 introns we call 4a, 4b and 4c were found. These three novel introns in combination with the ancestral introns 1 to 6, define three basic tetraspanin gene structures which have been conserved throughout the animal kingdom. Our phylogenomic approach also allows the estimation of the time at which the introns of the 33 human tetraspanin paralogs appeared, which in many cases coincides with the concomitant acquisition of new introns. On the other hand, we observed that new introns (introns other than 1-6, 4a, b and c were not randomly inserted into the tetraspanin gene structure. The region of tetraspanin genes corresponding to the small extracellular loop (SEL accounts for only 10.5% of the total sequence length but had 46% of the new animal intron insertions. CONCLUSIONS/SIGNIFICANCE: Our results indicate that tests of intron evolution are strengthened by the phylogenomic approach with

  4. Assessment of orthologous splicing isoforms in human and mouse orthologous genes

    Directory of Open Access Journals (Sweden)

    Horner David S

    2010-10-01

    Full Text Available Abstract Background Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level. Results As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns. Conclusions We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species

  5. A novel splicing mutation in the V2 vasopressin receptor

    DEFF Research Database (Denmark)

    Kamperis, Konstantinos; Siggaard, C; Herlin, Troels;

    2000-01-01

    In order to elucidate the molecular basis and the clinical characteristics of X-linked recessive nephrogenic diabetes insipidus (CNDI) in a kindred of Danish descent, we performed direct sequencing of the arginine vasopressin receptor 2 (AVPR2) gene in five members of the family, as well as...... clinical investigations comprising a fluid deprivation test and a 1-deamino-8-D-arginine-vasopressin (dDAVP) infusion test in the study subject and his mother. We found a highly unusual, novel, de novo 1447A-->C point mutation (gDNA), involving the invariable splice acceptor of the second intron of the...

  6. Lights, camera, action! Capturing the spliceosome and pre-mRNA splicing with single-molecule fluorescence microscopy.

    Science.gov (United States)

    DeHaven, Alexander C; Norden, Ian S; Hoskins, Aaron A

    2016-09-01

    The process of removing intronic sequences from a precursor to messenger RNA (pre-mRNA) to yield a mature mRNA transcript via splicing is an integral step in eukaryotic gene expression. Splicing is carried out by a cellular nanomachine called the spliceosome that is composed of RNA components and dozens of proteins. Despite decades of study, many fundamentals of spliceosome function have remained elusive. Recent developments in single-molecule fluorescence microscopy have afforded new tools to better probe the spliceosome and the complex, dynamic process of splicing by direct observation of single molecules. These cutting-edge technologies enable investigators to monitor the dynamics of specific splicing components, whole spliceosomes, and even cotranscriptional splicing within living cells. WIREs RNA 2016, 7:683-701. doi: 10.1002/wrna.1358 For further resources related to this article, please visit the WIREs website. PMID:27198613

  7. Polymorphism of the aryl-hydrocarbon receptor gene in intron 10 of human cancers

    Directory of Open Access Journals (Sweden)

    M. Rocas

    2011-11-01

    Full Text Available Polychlorinated dibenzo-p-dioxins (PCDDs and related halogenated aromatic hydrocarbons (e.g., PCDFs, often called "dioxins", are ubiquitously present environmental contaminants. Some of them, notably 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, are among the most toxic synthetic compounds known. The biological effects of dioxins are mediated via the aryl hydrocarbon receptor (AhR. Mutations in the AhR transactivation domain are linked to sensitivity to the acute lethality of TCDD. We present here a study of AhR gene polymorphism in normal and cancer human tissues affecting pre-mRNA splicing in the AhR gene-coding transactivation domain region (exon 10, intron 10, exon 11 region, previously shown to be associated with AhR dysfunction. We tested 126 pairs of normal and cancer tissue samples from liver, lung, stomach, kidney, mucous, breast, and pancreas of 49 males and 77 females (45-70 years of age. We used in vitro splicing assay, RT-PCR and sequencing methods. Our results showed that in an in vitro system it is possible to reconstitute cellular pre-mRNA splicing events. Tested cancer tissues did not contain mutations in the AhR transactivation domain region when the DNA sequences were compared with those from normal tissues. There were also no differences in AhR mRNA splice variants between normal and malignant breast tissues and no polymorphisms in the studied regions or cDNA.

  8. Polymorphism of the aryl-hydrocarbon receptor gene in intron 10 of human cancers.

    Science.gov (United States)

    Rocas, M; Jakubauskiene, E; Kanopka, A

    2011-11-01

    Polychlorinated dibenzo-p-dioxins (PCDDs) and related halogenated aromatic hydrocarbons (e.g., PCDFs), often called "dioxins", are ubiquitously present environmental contaminants. Some of them, notably 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are among the most toxic synthetic compounds known. The biological effects of dioxins are mediated via the aryl hydrocarbon receptor (AhR). Mutations in the AhR transactivation domain are linked to sensitivity to the acute lethality of TCDD. We present here a study of AhR gene polymorphism in normal and cancer human tissues affecting pre-mRNA splicing in the AhR gene-coding transactivation domain region (exon 10, intron 10, exon 11 region), previously shown to be associated with AhR dysfunction. We tested 126 pairs of normal and cancer tissue samples from liver, lung, stomach, kidney, mucous, breast, and pancreas of 49 males and 77 females (45-70 years of age). We used in vitro splicing assay, RT-PCR and sequencing methods. Our results showed that in an in vitro system it is possible to reconstitute cellular pre-mRNA splicing events. Tested cancer tissues did not contain mutations in the AhR transactivation domain region when the DNA sequences were compared with those from normal tissues. There were also no differences in AhR mRNA splice variants between normal and malignant breast tissues and no polymorphisms in the studied regions or cDNA. PMID:22052373

  9. Alternative splicing and muscular dystrophy

    OpenAIRE

    Pistoni, Mariaelena; Ghigna, Claudia; Gabellini, Davide

    2010-01-01

    Alternative splicing of pre-mRNAs is a major contributor to proteomic diversity and to the control of gene expression in higher eukaryotic cells. For this reasons, alternative splicing is tightly regulated in different tissues and developmental stages and its disruption can lead to a wide range of human disorders. The aim of this review is to focus on the relevance of alternative splicing for muscle function and muscle disease. We begin by giving a brief overview of alternative splicing, musc...

  10. Determinants of the Usage of Splice-Associated cis-Motifs Predict the Distribution of Human Pathogenic SNPs

    Science.gov (United States)

    Wu, XianMing; Hurst, Laurence D.

    2016-01-01

    Where in genes do pathogenic mutations tend to occur and does this provide clues as to the possible underlying mechanisms by which single nucleotide polymorphisms (SNPs) cause disease? As splice-disrupting mutations tend to occur predominantly at exon ends, known also to be hot spots of cis-exonic splice control elements, we examine the relationship between the relative density of such exonic cis-motifs and pathogenic SNPs. In particular, we focus on the intragene distribution of exonic splicing enhancers (ESE) and the covariance between them and disease-associated SNPs. In addition to showing that disease-causing genes tend to be genes with a high intron density, consistent with missplicing, five factors established as trends in ESE usage, are considered: relative position in exons, relative position in genes, flanking intron size, splice sites usage, and phase. We find that more than 76% of pathogenic SNPs are within 3–69 bp of exon ends where ESEs generally reside, this being 13% more than expected. Overall from enrichment of pathogenic SNPs at exon ends, we estimate that approximately 20–45% of SNPs affect splicing. Importantly, we find that within genes pathogenic SNPs tend to occur in splicing-relevant regions with low ESE density: they are found to occur preferentially in the terminal half of genes, in exons flanked by short introns and at the ends of phase (0,0) exons with 3′ non-“AGgt” splice site. We suggest the concept of the “fragile” exon, one home to pathogenic SNPs owing to its vulnerability to splice disruption owing to low ESE density. PMID:26545919

  11. Microbial and Natural Metabolites That Inhibit Splicing: A Powerful Alternative for Cancer Treatment.

    Science.gov (United States)

    Martínez-Montiel, Nancy; Rosas-Murrieta, Nora Hilda; Martínez-Montiel, Mónica; Gaspariano-Cholula, Mayra Patricia; Martínez-Contreras, Rebeca D

    2016-01-01

    In eukaryotes, genes are frequently interrupted with noncoding sequences named introns. Alternative splicing is a nuclear mechanism by which these introns are removed and flanking coding regions named exons are joined together to generate a message that will be translated in the cytoplasm. This mechanism is catalyzed by a complex machinery known as the spliceosome, which is conformed by more than 300 proteins and ribonucleoproteins that activate and regulate the precision of gene expression when assembled. It has been proposed that several genetic diseases are related to defects in the splicing process, including cancer. For this reason, natural products that show the ability to regulate splicing have attracted enormous attention due to its potential use for cancer treatment. Some microbial metabolites have shown the ability to inhibit gene splicing and the molecular mechanism responsible for this inhibition is being studied for future applications. Here, we summarize the main types of natural products that have been characterized as splicing inhibitors, the recent advances regarding molecular and cellular effects related to these molecules, and the applications reported so far in cancer therapeutics. PMID:27610372

  12. Thermodynamic modeling of donor splice site recognition in pre-mRNA

    Science.gov (United States)

    Garland, Jeffrey A.; Aalberts, Daniel P.

    2004-04-01

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

  13. Modulation of p53β and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response

    OpenAIRE

    Marcel, V; Fernandes, K; Terrier, O; LANE, D. P.; Bourdon, J-C

    2014-01-01

    In addition to the tumor suppressor p53 protein, also termed p53α, the TP53 gene produces p53β and p53γ through alternative splicing of exons 9β and 9γ located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53β and p53γ at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdo...

  14. The pathogenicity of splicing defects: mechanistic insights into pre-mRNA processing inform novel therapeutic approaches.

    Science.gov (United States)

    Daguenet, Elisabeth; Dujardin, Gwendal; Valcárcel, Juan

    2015-12-01

    Removal of introns from pre-mRNA precursors (pre-mRNA splicing) is a necessary step for the expression of most genes in multicellular organisms, and alternative patterns of intron removal diversify and regulate the output of genomic information. Mutation or natural variation in pre-mRNA sequences, as well as in spliceosomal components and regulatory factors, has been implicated in the etiology and progression of numerous pathologies. These range from monogenic to multifactorial genetic diseases, including metabolic syndromes, muscular dystrophies, neurodegenerative and cardiovascular diseases, and cancer. Understanding the molecular mechanisms associated with splicing-related pathologies can provide key insights into the normal function and physiological context of the complex splicing machinery and establish sound basis for novel therapeutic approaches. PMID:26566663

  15. Group I introns and associated homing endonuclease genes reveals a clinal structure for Porphyra spiralis var. amplifolia (Bangiales, Rhodophyta along the Eastern coast of South America

    Directory of Open Access Journals (Sweden)

    Matioli Sergio R

    2008-11-01

    Full Text Available Abstract Background Group I introns are found in the nuclear small subunit ribosomal RNA gene (SSU rDNA of some species of the genus Porphyra (Bangiales, Rhodophyta. Size polymorphisms in group I introns has been interpreted as the result of the degeneration of homing endonuclease genes (HEG inserted in peripheral loops of intron paired elements. In this study, intron size polymorphisms were characterized for different Porphyra spiralis var. amplifolia (PSA populations on the Southern Brazilian coast, and were used to infer genetic relationships and genetic structure of these PSA populations, in addition to cox2-3 and rbcL-S regions. Introns of different sizes were tested qualitatively for in vitro self-splicing. Results Five intron size polymorphisms within 17 haplotypes were obtained from 80 individuals representing eight localities along the distribution of PSA in the Eastern coast of South America. In order to infer genetic structure and genetic relationships of PSA, these polymorphisms and haplotypes were used as markers for pairwise Fst analyses, Mantel's test and median joining network. The five cox2-3 haplotypes and the unique rbcL-S haplotype were used as markers for summary statistics, neutrality tests Tajima's D and Fu's Fs and for median joining network analyses. An event of demographic expansion from a population with low effective number, followed by a pattern of isolation by distance was obtained for PSA populations with the three analyses. In vitro experiments have shown that introns of different lengths were able to self-splice from pre-RNA transcripts. Conclusion The findings indicated that degenerated HEGs are reminiscent of the presence of a full-length and functional HEG, once fixed for PSA populations. The cline of HEG degeneration determined the pattern of isolation by distance. Analyses with the other markers indicated an event of demographic expansion from a population with low effective number. The different degrees of

  16. Where splicing joins chromatin

    Czech Academy of Sciences Publication Activity Database

    Hnilicová, Jarmila; Staněk, David

    2011-01-01

    Roč. 2, č. 3 (2011), s. 182-188. ISSN 1949-1034 R&D Projects: GA ČR GAP305/10/0424; GA AV ČR KAN200520801 Institutional research plan: CEZ:AV0Z50520514 Keywords : chromatin * exon * alternative splicing * transcription * snRNP Subject RIV: EB - Genetics ; Molecular Biology

  17. Functional and splicing defect analysis of 23 ACVRL1 mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia.

    Directory of Open Access Journals (Sweden)

    Ferdos Alaa El Din

    Full Text Available Hereditary Hemorrhagic Telangiectasia syndrome (HHT or Rendu-Osler-Weber (ROW syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG and activin receptor-like kinase 1 (ACVRL1or ALK1 genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1, the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7 was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of ACVRL1 induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations.

  18. H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast

    LENUS (Irish Health Repository)

    Shieh, Grace S.

    2011-12-22

    Abstract Background The packaging of DNA into chromatin regulates transcription from initiation through 3\\' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA. Results Here we provide evidence that H2B monoubiquitylation (H2BK123ub1) marks introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3), which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an htb-K123R mutation, leads to synthetic lethality. Conclusion These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.

  19. Evolution of rbcL group IA introns and intron open reading frames within the colonial Volvocales (Chlorophyceae).

    Science.gov (United States)

    Nozaki, Hisayoshi; Takahara, Manabu; Nakazawa, Atsushi; Kita, Yoko; Yamada, Takashi; Takano, Hiroyoshi; Kawano, Shigeyuki; Kato, Masahiro

    2002-06-01

    Mobile group I introns sometimes contain an open reading frame (ORF) possibly encoding a site-specific DNA endonuclease. However, previous phylogenetic studies have not clearly deduced the evolutionary roles of the group I intron ORFs. In this paper, we examined the phylogeny of group IA2 introns inserted in the position identical to that of the chloroplast-encoded rbcL coding region (rbcL-462 introns) and their ORFs from 13 strains of five genera (Volvox, Pleodorina, Volvulina, Astrephomene, and Gonium) of the colonial Volvocales (Chlorophyceae) and a related unicellular green alga, Vitreochlamys. The rbcL-462 introns contained an intact or degenerate ORF of various sizes except for the Gonium multicoccum rbcL-462 intron. Partial amino acid sequences of some rbcL-462 intron ORFs exhibited possible homology to the endo/excinuclease amino acid terminal domain. The distribution of the rbcL-462 introns is sporadic in the phylogenetic trees of the colonial Volvocales based on the five chloroplast exon sequences (6021 bp). Phylogenetic analyses of the conserved intron sequences resolved that the G. multicoccum rbcL-462 intron had a phylogenetic position separate from those of other colonial volvocalean rbcL-462 introns, indicating the recent horizontal transmission of the intron in the G. multicoccum lineage. However, the combined data set from conserved intron sequences and ORFs from most of the rbcL-462 introns resolved robust phylogenetic relationships of the introns that were consistent with those of the host organisms. Therefore, most of the extant rbcL-462 introns may have been vertically inherited from the common ancestor of their host organisms, whereas such introns may have been lost in other lineages during evolution of the colonial Volvocales. In addition, apparently higher synonymous substitutions than nonsynonymous substitutions in the rbcL-462 intron ORFs indicated that the ORFs might evolve under functional constraint, which could result in homing of the

  20. Cloning and Alternative Splicing Analysis of Bombyx mori Transformer-2 Gene using Silkworm EST Database

    Institute of Scientific and Technical Information of China (English)

    Bao-Long NIU; Zhi-Qi MENG; Yue-Zhi TAO; Shun-Lin LU; Hong-Biao WENG; Li-Hua HE; Wei-Feng SHEN

    2005-01-01

    We have identified Bombyx mori transformer-2 gene (Bmtra-2) cDNA by blasting the EST database of B. mori. It was expressed in the whole life of the male and female silkworm and was observed as a band of 1.3 kb by Northern blot analysis. By comparing corresponding ESTs to the Bmtra-2 DNA sequence,it was revealed that there were eight exons and seven introns, and all splice sites of exons/introns conformed to the GT/AG rule. Bmtra-2 pre-mRNA can produce multiple mRNAs encoding six distinct isoforms of BmTRA-2 protein using an alternative splicing pathway during processing. Six types of Bmtra-2 cDNA clones were identified by reverse transcription-polymerase chain reaction. All isoforms of BmTRA-2 protein contain two arginine/serine-rich domains and one RNA recognition motif, showing striking organizational similarity to Drosophila TRA-2 proteins.

  1. Spliced leader trans-splicing in the nematode Trichinella spiralis uses highly polymorphic, noncanonical spliced leaders

    OpenAIRE

    Pettitt, Jonathan; Müller, Berndt; Stansfield, Ian; Connolly, Bernadette

    2008-01-01

    The trans-splicing of short spliced leader (SL) RNAs onto the 5′ ends of mRNAs occurs in a diverse range of taxa. In nematodes, all species so far characterized utilize a characteristic, conserved spliced leader, SL1, as well as variants that are employed in the resolution of operons. Here we report the identification of spliced leader trans-splicing in the basal nematode Trichinella spiralis, and show that this nematode does not possess a canonical SL1, but rather has at least 15 distinct sp...

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

    Science.gov (United States)

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

    2014-03-01

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

  3. The Exon Junction Complex Controls the Efficient and Faithful Splicing of a Subset of Transcripts Involved in Mitotic Cell-Cycle Progression.

    Science.gov (United States)

    Fukumura, Kazuhiro; Wakabayashi, Shunichi; Kataoka, Naoyuki; Sakamoto, Hiroshi; Suzuki, Yutaka; Nakai, Kenta; Mayeda, Akila; Inoue, Kunio

    2016-01-01

    The exon junction complex (EJC) that is deposited onto spliced mRNAs upstream of exon-exon junctions plays important roles in multiple post-splicing gene expression events, such as mRNA export, surveillance, localization, and translation. However, a direct role for the human EJC in pre-mRNA splicing has not been fully understood. Using HeLa cells, we depleted one of the EJC core components, Y14, and the resulting transcriptome was analyzed by deep sequencing (RNA-Seq) and confirmed by RT-PCR. We found that Y14 is required for efficient and faithful splicing of a group of transcripts that is enriched in short intron-containing genes involved in mitotic cell-cycle progression. Tethering of EJC core components (Y14, eIF4AIII or MAGOH) to a model reporter pre-mRNA harboring a short intron showed that these core components are prerequisites for the splicing activation. Taken together, we conclude that the EJC core assembled on pre-mRNA is critical for efficient and faithful splicing of a specific subset of short introns in mitotic cell cycle-related genes. PMID:27490541

  4. Conservation and sex-specific splicing of the doublesex gene in the economically important pest species Lucilia cuprina

    Indian Academy of Sciences (India)

    Carolina Concha; Fang Li; Maxwell J. Scott

    2010-09-01

    Genetic control of sex determination in insects has been best characterized in Drosophila melanogaster, where the master gene Sxl codes for RNA that is sex specifically spliced to produce a functional protein only in females. SXL regulates the sex-specific splicing of transformer (tra) RNA which, in turn, regulates the splicing of dsx RNA to produce functional male and female proteins. In the Australian sheep blowfly (Lucilia cuprina), the tra gene (Lctra) is required for female development and Lctra transcripts are sex-specifically spliced such that only female Lctra mRNA codes for functional protein. In males, a factor encoded by the Y-linked male determining gene is thought to prevent the female-mode of splicing of Lctra RNA. To further our understanding of the sex determination regulatory hierarchy in L. cuprina, we have isolated the dsx gene (Lcdsx) from this species. We found that the Lcdsx transcripts are sex-specifically spliced in a similar manner as their counterparts in D. melanogaster, housefly and tephritids. The LcDSX proteins are well conserved and the male form of DSX contains a motif encoded by a male-specific exon that is within the female-specific intron. This intron/exon arrangement had previously been found only in the housefly dsx gene, suggesting this may be a unique feature of dsx genes of Calyptratae species.

  5. Tuning of alternative splicing--switch from proto-oncogene to tumor suppressor.

    Science.gov (United States)

    Shchelkunova, Aleksandra; Ermolinsky, Boris; Boyle, Meghan; Mendez, Ivan; Lehker, Michael; Martirosyan, Karen S; Kazansky, Alexander V

    2013-01-01

    STAT5B, a specific member of the STAT family, is intimately associated with prostate tumor progression. While the full form of STAT5B is thought to promote tumor progression, a naturally occurring truncated isoform acts as a tumor suppressor. We previously demonstrated that truncated STAT5 is generated by insertion of an alternatively spliced exon and results in the introduction of an early termination codon. Present approaches targeting STAT proteins based on inhibition of functional domains of STAT's, such as DNA-binding, cooperative binding (protein-protein interaction), dimerization and phosphorylation will halt the action of the entire gene, both the proto-oncogenic and tumor suppressor functions of Stat5B. In this report we develop a new approach aimed at inhibiting the expression of full-length STAT5B (a proto-oncogene) while simultaneously enhancing the expression of STAT5∆B (a tumor suppressor). We have demonstrated the feasibility of using steric-blocking splice-switching oligonucleotides (SSOs) with a complimentary sequence to the targeted exon-intron boundary to enhance alternative intron/exon retention (up to 10%). The functional effect of the intron/exon proportional tuning was validated by cell proliferation and clonogenic assays. The new scheme applies specific steric-blocking splice-switching oligonucleotides and opens an opportunity for anti-tumor treatment as well as for the alteration of functional abilities of other STAT proteins. PMID:23289016

  6. Tuning of Alternative Splicing - Switch From Proto-Oncogene to Tumor Suppressor

    Directory of Open Access Journals (Sweden)

    Aleksandra Shchelkunova, Boris Ermolinsky, Meghan Boyle, Ivan Mendez, Michael Lehker, Karen S. Martirosyan, Alexander V. Kazansky

    2013-01-01

    Full Text Available STAT5B, a specific member of the STAT family, is intimately associated with prostate tumor progression. While the full form of STAT5B is thought to promote tumor progression, a naturally occurring truncated isoform acts as a tumor suppressor. We previously demonstrated that truncated STAT5 is generated by insertion of an alternatively spliced exon and results in the introduction of an early termination codon. Present approaches targeting STAT proteins based on inhibition of functional domains of STAT's, such as DNA-binding, cooperative binding (protein-protein interaction, dimerization and phosphorylation will halt the action of the entire gene, both the proto-oncogenic and tumor suppressor functions of Stat5B.In this report we develop a new approach aimed at inhibiting the expression of full-length STAT5B (a proto-oncogene while simultaneously enhancing the expression of STAT5∆B (a tumor suppressor. We have demonstrated the feasibility of using steric-blocking splice-switching oligonucleotides (SSOs with a complimentary sequence to the targeted exon-intron boundary to enhance alternative intron/exon retention (up to 10%. The functional effect of the intron/exon proportional tuning was validated by cell proliferation and clonogenic assays. The new scheme applies specific steric-blocking splice-switching oligonucleotides and opens an opportunity for anti-tumor treatment as well as for the alteration of functional abilities of other STAT proteins.

  7. Tuning of Alternative Splicing - Switch From Proto-Oncogene to Tumor Suppressor

    Science.gov (United States)

    Shchelkunova, Aleksandra; Ermolinsky, Boris; Boyle, Meghan; Mendez, Ivan; Lehker, Michael; Martirosyan, Karen S.; Kazansky, Alexander V.

    2013-01-01

    STAT5B, a specific member of the STAT family, is intimately associated with prostate tumor progression. While the full form of STAT5B is thought to promote tumor progression, a naturally occurring truncated isoform acts as a tumor suppressor. We previously demonstrated that truncated STAT5 is generated by insertion of an alternatively spliced exon and results in the introduction of an early termination codon. Present approaches targeting STAT proteins based on inhibition of functional domains of STAT's, such as DNA-binding, cooperative binding (protein-protein interaction), dimerization and phosphorylation will halt the action of the entire gene, both the proto-oncogenic and tumor suppressor functions of Stat5B. In this report we develop a new approach aimed at inhibiting the expression of full-length STAT5B (a proto-oncogene) while simultaneously enhancing the expression of STAT5∆B (a tumor suppressor). We have demonstrated the feasibility of using steric-blocking splice-switching oligonucleotides (SSOs) with a complimentary sequence to the targeted exon-intron boundary to enhance alternative intron/exon retention (up to 10%). The functional effect of the intron/exon proportional tuning was validated by cell proliferation and clonogenic assays. The new scheme applies specific steric-blocking splice-switching oligonucleotides and opens an opportunity for anti-tumor treatment as well as for the alteration of functional abilities of other STAT proteins. PMID:23289016

  8. The splicing fate of plant SPO11 genes

    Directory of Open Access Journals (Sweden)

    Thorben eSprink

    2014-05-01

    Full Text Available Towards the global understanding of plant meiosis, it seems to be essential to decipher why all as yet sequenced plants need or at least encode for two different meiotic SPO11 genes. This is in contrast to mammals and fungi, where only one SPO11 is present. Both SPO11 in plants are essential for the initiation of double strand breaks (DSBs during the meiotic prophase. In nearly all eukaryotic organisms DSB induction by SPO11 leads to meiotic DSB repair, thereby ensuring the formation of a necessary number of crossovers (CO as physical connections between the allelic chromosomes. We aim to investigate the specific functions and evolution of both SPO11 genes in land plants. Therefore, we identified and cloned the respective orthologous genes from Brassica rapa, Carica papaya, Oryza sativa and Physcomitrella patens. In parallel we determined the full length cDNA sequences of SPO11-1 and -2 from all of these plants by RT-PCR. During these experiments we observed that the analyzed plants exhibit a pattern of aberrant splicing products of both SPO11 mRNAs. Such an aberrant splicing has previously been described for Arabidopsis and therefore seems to be conserved throughout evolution. Most of the splicing forms of SPO11-1 and -2 seem to be non functional as they either showed intron retention or shortened exons accompanied by a frameshift leading to premature termination codons (PTCs in most cases. Nevertheless, we could detect one putative functional alternatively spliced mRNA for SPO11-1 and -2 each, indicating that splicing of SPO11 does not depend only on the gene sequence but also on the plant species and that it might play a regulatory role.

  9. A unique, consistent identifier for alternatively spliced transcript variants.

    Directory of Open Access Journals (Sweden)

    Alberto Riva

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

  10. Intronic deletions of tva receptor gene decrease the susceptibility to infection by avian sarcoma and leukosis virus subgroup A.

    Science.gov (United States)

    Chen, Weiguo; Liu, Yang; Li, Hongxing; Chang, Shuang; Shu, Dingming; Zhang, Huanmin; Chen, Feng; Xie, Qingmei

    2015-01-01

    The group of avian sarcoma and leukosis virus (ASLV) in chickens contains six highly related subgroups, A to E and J. Four genetic loci, tva, tvb, tvc and tvj, encode for corresponding receptors that determine the susceptibility to the ASLV subgroups. The prevalence of ASLV in hosts may have imposed strong selection pressure toward resistance to ASLV infection, and the resistant alleles in all four receptor genes have been identified. In this study, two new alleles of the tva receptor gene, tva(r5) and tva(r6), with similar intronic deletions were identified in Chinese commercial broilers. These natural mutations delete the deduced branch point signal within the first intron, disrupting mRNA splicing of the tva receptor gene and leading to the retention of intron 1 and introduction of premature TGA stop codons in both the longer and shorter tva isoforms. As a result, decreased susceptibility to subgroup A ASLV in vitro and in vivo was observed in the subsequent analysis. In addition, we identified two groups of heterozygous allele pairs which exhibited quantitative differences in host susceptibility to ASLV-A. This study demonstrated that defective splicing of the tva receptor gene can confer genetic resistance to ASLV subgroup A in the host. PMID:25873518

  11. Is the human dystrophin gene's intron structure related to its intron instability?

    Institute of Scientific and Technical Information of China (English)

    盛文利; 陈江瑛; 朱良付; 刘焯霖

    2003-01-01

    Objective To study the human dystrophin gene molecular deletion mechanism, we analyzed breakpoint regions within junction fragments of deletion-type patients and investigated whether the dystrophin gene's intron structure might be related to intron instability.Methods Junction fragments corresponding to exon 46 and 51 deletions were cloned. The breakpoint regions were sequenced, and the features of introns with available Genebank sequences were analyzed.Results An analysis of junction fragment sequences corresponding to exon 46 and 51 deletions showed that all 5' and 3' breakpoints are located within repeat sequences. No small insertions, small deletions, or point mutations are located near the breakpoint junctions. By analyzing the secondary structure of the junction fragments, we demonstrated that all junction fragment breakpoints are located in non-matching regions of single-stranded hairpin loops. A high concentration of repetitive elements is found to be a key feature of many dystrophin introns. In total, 34.8% of the overall dystrophin intron sequences is composed of repeat sequences.Conclusion Repeat elements in many dystrophin gene introns are the key to their structural bases and reflect intron instability. As a result of the primary DNA sequences, single-stranded hairpin loops form, increasing the instability of the gene, and forming the base for breaks in the DNA. The formation of the single-stranded hairpins can result in reattachment of two different breakpoints, producing a deletion.

  12. Alternative splicing modulated by genetic variants demonstrates accelerated evolution regulated by highly conserved proteins.

    Science.gov (United States)

    Hsiao, Yun-Hua Esther; Bahn, Jae Hoon; Lin, Xianzhi; Chan, Tak-Ming; Wang, Rena; Xiao, Xinshu

    2016-04-01

    Identification of functional genetic variants and elucidation of their regulatory mechanisms represent significant challenges of the post-genomic era. A poorly understood topic is the involvement of genetic variants in mediating post-transcriptional RNA processing, including alternative splicing. Thus far, little is known about the genomic, evolutionary, and regulatory features of genetically modulated alternative splicing (GMAS). Here, we systematically identified intronic tag variants for genetic modulation of alternative splicing using RNA-seq data specific to cellular compartments. Combined with our previous method that identifies exonic tags for GMAS, this study yielded 622 GMAS exons. We observed that GMAS events are highly cell type independent, indicating that splicing-altering genetic variants could have widespread function across cell types. Interestingly, GMAS genes, exons, and single-nucleotide variants (SNVs) all demonstrated positive selection or accelerated evolution in primates. We predicted that GMAS SNVs often alter binding of splicing factors, with SRSF1 affecting the most GMAS events and demonstrating global allelic binding bias. However, in contrast to their GMAS targets, the predicted splicing factors are more conserved than expected, suggesting thatcis-regulatory variation is the major driving force of splicing evolution. Moreover, GMAS-related splicing factors had stronger consensus motifs than expected, consistent with their susceptibility to SNV disruption. Intriguingly, GMAS SNVs in general do not alter the strongest consensus position of the splicing factor motif, except the more than 100 GMAS SNVs in linkage disequilibrium with polymorphisms reported by genome-wide association studies. Our study reports many GMAS events and enables a better understanding of the evolutionary and regulatory features of this phenomenon. PMID:26888265

  13. The neurogenetics of alternative splicing

    OpenAIRE

    Vuong, CK; Black, DL; S. Zheng

    2016-01-01

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

  14. SL1 trans-splicing specified by AU-rich synthetic RNA inserted at the 5' end of Caenorhabditis elegans pre-mRNA.

    OpenAIRE

    Conrad, R; Lea, K; Blumenthal, T

    1995-01-01

    In Caenorhabditis elegans, pre-mRNAs of many genes are trans-spliced to one of two spliced leaders, SL1 or SL2. Some of those that receive exclusively SL1 have been characterized as having at their 5' ends outrons, AU-rich sequences similar to introns followed by conventional 3' splice sites. Comparison of outrons from many different SL1-specific C. elegans genes has not revealed the presence of any consensus sequence that might encode SL1-specificity. In order to determine what parameters in...

  15. Genetic enhancement of RNA-processing defects by a dominant mutation in B52, the Drosophila gene for an SR protein splicing factor.

    OpenAIRE

    Peng, X; Mount, S M

    1995-01-01

    SR proteins are essential for pre-mRNA splicing in vitro, act early in the splicing pathway, and can influence alternative splice site choice. Here we describe the isolation of both dominant and loss-of-function alleles of B52, the gene for a Drosophila SR protein. The allele B52ED was identified as a dominant second-site enhancer of white-apricot (wa), a retrotransposon insertion in the second intron of the eye pigmentation gene white with a complex RNA-processing defect. B52ED also exaggera...

  16. The tRNATyr multigene family of Triticum aestivum: genome organization, sequence analyses and maturation of intron-containing pre-tRNAs in wheat germ extract.

    Science.gov (United States)

    Arends, S; Kraus, J; Beier, H

    1996-04-22

    Southern analysis of Triticum DNA has revealed that nuclear tRNATyr genes are dispersed at a minimum of 16 loci in the genome. We have isolated six independent tRNATyr genes from a Triticum aestivum library in addition to three known members of the Triticum tRNATyr family. Four of the sequenced tRNATyr genes code for Triticum tRNA Tyr and two code for tRNA2Tyr. Three genes encode tRNAsTyr which carry one or two nucleotide substitutions as compared to the conventional genes. The nine Triticum tRNATyr genes possess highly conserved intron sequences ranging in size from 12 to 14 nucleotides. A common secondary intron structure with the 5' and 3' splice site loops separated by five base pairs can be formed by all pre-tRNAs Tyr which are efficiently spliced in the homologous wheat germ extract. PMID:8617358

  17. SNW1 enables sister chromatid cohesion by mediating the splicing of sororin and APC2 pre-mRNAs

    Science.gov (United States)

    van der Lelij, Petra; Stocsits, Roman R; Ladurner, Rene; Petzold, Georg; Kreidl, Emanuel; Koch, Birgit; Schmitz, Julia; Neumann, Beate; Ellenberg, Jan; Peters, Jan-Michael

    2014-01-01

    Although splicing is essential for the expression of most eukaryotic genes, inactivation of splicing factors causes specific defects in mitosis. The molecular cause of this defect is unknown. Here, we show that the spliceosome subunits SNW1 and PRPF8 are essential for sister chromatid cohesion in human cells. A transcriptome-wide analysis revealed that SNW1 or PRPF8 depletion affects the splicing of specific introns in a subset of pre-mRNAs, including pre-mRNAs encoding the cohesion protein sororin and the APC/C subunit APC2. SNW1 depletion causes cohesion defects predominantly by reducing sororin levels, which causes destabilisation of cohesin on DNA. SNW1 depletion also reduces APC/C activity and contributes to cohesion defects indirectly by delaying mitosis and causing “cohesion fatigue”. Simultaneous expression of sororin and APC2 from intron-less cDNAs restores cohesion in SNW1-depleted cells. These results indicate that the spliceosome is required for mitosis because it enables expression of genes essential for cohesion. Our transcriptome-wide identification of retained introns in SNW1- and PRPF8-depleted cells may help to understand the aetiology of diseases associated with splicing defects, such as retinosa pigmentosum and cancer. PMID:25257309

  18. Transcript specificity in yeast pre-mRNA splicing revealed by mutations in core spliceosomal components.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Pleiss

    2007-04-01

    Full Text Available Appropriate expression of most eukaryotic genes requires the removal of introns from their pre-messenger RNAs (pre-mRNAs, a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well.

  19. 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

    2014-08-01

    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

  20. Comparison of the unlabeled and labeled pre-mRNA splicing assays in vitro

    Institute of Scientific and Technical Information of China (English)

    TIAN XU BU; JING XIN HONG; ZHI YAO; JIE YANG

    2006-01-01

    Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome that catalyzes the removal of non-coding intron sequences to ligate exons into mature mRNA prior to transport and translation. The purpose of our study is to explore whether the in vitro unlabeled pre-mRNA splicing assay could be performed as an alternative method of splicing reaction other than the radiolabeled one. Two different splicing methods in vitro, 32P labeled and unlabeled pre-mRNA as the substrates in the reaction, were investigated. The radiolabeled products were visualized by autoradiography while the unlabeled products were observed by Ethidium Bromide (EB)staining. As a result, although there are more unspecific bands in the EB staining assay than 32P labeled one, the RNA products of in vitro splicing could be observed clearly. This suggests that the unlabeled pre-mRNA splicing assay can be an optional substitution for the isotope-labeled assay.

  1. Exon-centric regulation of pyruvate kinase M alternative splicing via mutually exclusive exons

    Institute of Scientific and Technical Information of China (English)

    Zhenxun Wang; Deblina Chatterjee; Hyun Yong Jeon; Martin Akerman; Matthew G. Vander Heiden; Lewis C. Cantley; Adrian R. Krainer

    2012-01-01

    Alternative splicing of the pyruvate kinase M gene (PK-M) can generate the M2 isoform and promote aerobic glycolysis and tumor growth.However,the cancer-specific alternative splicing regulation of PK-M is not completely understood.Here,we demonstrate that PK-M is regulated by reciprocal affects on the mutually exclusive exons 9 and 10,such that exon 9 is repressed and exon 10 is activated in cancer cells.Strikingly,exonic,rather than intronic,cis-elements are key determinants ef PK-M splicing isoform ratios.Using a systematic sub-exonic duplication approach,we identify a potent exonlc splicing enhancer in exon 10,which differs from its homologous counterpart in exon 9 by only two nucleotides.We identify SRSF3 as one of the cognate factors,and show that this serine/arginine-rich protein activates exon 10 and mediates changes in glucose metabolism.These findings provide mechanistic insights into the complex regulation of alternative splicing of a key regulator of the Warburg effect,and also have implications for other genes with a similar pattern of alternative splicing.

  2. Fusion splicing of silicon optical fibres

    OpenAIRE

    Xiao, L.M.; Healy, N; Gibson, U.; Hawkins, T.; Jones, M.; Ballato, J; A. C. Peacock

    2015-01-01

    The first splicing experiments between silicon optical fibres (SOFs) and conventional fibres are investigated. An optimized fusion splicing approach for a polycrystalline SOF is demonstrated and the material properties after splicing are characterized.

  3. Involvement of Alternative Splicing in Barley Seed Germination.

    Directory of Open Access Journals (Sweden)

    Qisen Zhang

    Full Text Available Seed germination activates many new biological processes including DNA, membrane and mitochondrial repairs and requires active protein synthesis and sufficient energy supply. Alternative splicing (AS regulates many cellular processes including cell differentiation and environmental adaptations. However, limited information is available on the regulation of seed germination at post-transcriptional levels. We have conducted RNA-sequencing experiments to dissect AS events in barley seed germination. We identified between 552 and 669 common AS transcripts in germinating barley embryos from four barley varieties (Hordeum vulgare L. Bass, Baudin, Harrington and Stirling. Alternative 3' splicing (34%-45%, intron retention (32%-34% and alternative 5' splicing (16%-21% were three major AS events in germinating embryos. The AS transcripts were predominantly mapped onto ribosome, RNA transport machineries, spliceosome, plant hormone signal transduction, glycolysis, sugar and carbon metabolism pathways. Transcripts of these genes were also very abundant in the early stage of seed germination. Correlation analysis of gene expression showed that AS hormone responsive transcripts could also be co-expressed with genes responsible for protein biosynthesis and sugar metabolisms. Our RNA-sequencing data revealed that AS could play important roles in barley seed germination.

  4. The functional consequences of intron retention

    DEFF Research Database (Denmark)

    Ge, Ying; Porse, Bo T

    2013-01-01

    The explosion in sequencing technologies has provided us with an instrument to describe mammalian transcriptomes at unprecedented depths. This has revealed that alternative splicing is used extensively not only to generate protein diversity, but also as a means to regulate gene expression post......). Here, we highlight how IR and, more broadly, alternative splicing coupled to NMD (AS-NMD) can be used to regulate gene expression and how this is deregulated in disease. We suggest that the importance of AS-NMD is not restricted to the haematopoietic system but that it plays a prominent role in other...

  5. Extensive intron gain in the ancestor of placental mammals

    Directory of Open Access Journals (Sweden)

    Kordiš Dušan

    2011-11-01

    Full Text Available Abstract Background Genome-wide studies of intron dynamics in mammalian orthologous genes have found convincing evidence for loss of introns but very little for intron turnover. Similarly, large-scale analysis of intron dynamics in a few vertebrate genomes has identified only intron losses and no gains, indicating that intron gain is an extremely rare event in vertebrate evolution. These studies suggest that the intron-rich genomes of vertebrates do not allow intron gain. The aim of this study was to search for evidence of de novo intron gain in domesticated genes from an analysis of their exon/intron structures. Results A phylogenomic approach has been used to analyse all domesticated genes in mammals and chordates that originated from the coding parts of transposable elements. Gain of introns in domesticated genes has been reconstructed on well established mammalian, vertebrate and chordate phylogenies, and examined as to where and when the gain events occurred. The locations, sizes and amounts of de novo introns gained in the domesticated genes during the evolution of mammals and chordates has been analyzed. A significant amount of intron gain was found only in domesticated genes of placental mammals, where more than 70 cases were identified. De novo gained introns show clear positional bias, since they are distributed mainly in 5' UTR and coding regions, while 3' UTR introns are very rare. In the coding regions of some domesticated genes up to 8 de novo gained introns have been found. Intron densities in Eutheria-specific domesticated genes and in older domesticated genes that originated early in vertebrates are lower than those for normal mammalian and vertebrate genes. Surprisingly, the majority of intron gains have occurred in the ancestor of placentals. Conclusions This study provides the first evidence for numerous intron gains in the ancestor of placental mammals and demonstrates that adequate taxon sampling is crucial for

  6. Pre-mRNA splicing within an assembled yeast spliceosome requires an RNA-dependent ATPase and ATP hydrolysis.

    OpenAIRE

    Kim, S. H.; Lin, R J

    1993-01-01

    Unlike autocatalyzed self-splicing of group I or group II introns, the removal of pre-mRNA introns in vitro occurs in the spliceosome. The spliceosome is a multicomponent complex composed of pre-mRNA, small nuclear ribonucleoprotein particles, and protein factors. ATP is required for the assembly of the spliceosome and both transesterification reactions. An RNA-dependent ATPase, the product of the yeast PRP2 gene, has been shown to be involved in the first transesterification of pre-mRNA spli...

  7. Genes of nuclear encoded mitochondrial proteins: evidence for a variant of the 3' splice-site consensus sequence.

    OpenAIRE

    Juretić, N; Jaussi, R; Mattes, U; Christen, P

    1987-01-01

    The introns of animal nuclear genes and of viral genes encoding protein possess at their 3' splice-site the consensus sequence (CT)11NTCAG (Mount, S.M. (1982) Nucl. Acids Res. 10, 459-472; Green, M.R. (1986) Ann. Rev. Genet. 20, 671-708). However, the total 39 introns of the 5 imported mitochondrial proteins of higher eucaryotes whose gene structure has been determined to date show a predominance of 44% for base T at position -4. Apparently, a variant consensus sequence, i.e. (CT)11TTCAG, cha...

  8. A five' splice-region G → C mutation in exon 1 of the human β-globin gene inhibits pre-mRNA splicing: A mechanism for β+-thalassemia

    International Nuclear Information System (INIS)

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

  9. The RNA-binding profile of Acinus, a peripheral component of the exon junction complex, reveals its role in splicing regulation.

    Science.gov (United States)

    Rodor, Julie; Pan, Qun; Blencowe, Benjamin J; Eyras, Eduardo; Cáceres, Javier F

    2016-09-01

    Acinus (apoptotic chromatin condensation inducer in the nucleus) is an RNA-binding protein (RBP) originally identified for its role in apoptosis. It was later found to be an auxiliary component of the exon junction complex (EJC), which is deposited at exon junctions as a consequence of pre-mRNA splicing. To uncover the cellular functions of Acinus and investigate its role in splicing, we mapped its endogenous RNA targets using the cross-linking immunoprecipitation protocol (iCLIP). We observed that Acinus binds to pre-mRNAs, associating specifically to a subset of suboptimal introns, but also to spliced mRNAs. We also confirmed the presence of Acinus as a peripheral factor of the EJC. RNA-seq was used to investigate changes in gene expression and alternative splicing following siRNA-mediated depletion of Acinus in HeLa cells. This analysis revealed that Acinus is preferentially required for the inclusion of specific alternative cassette exons and also controls the faithful splicing of a subset of introns. Moreover, a large number of splicing changes can be related to Acinus binding, suggesting a direct role of Acinus in exon and intron definition. In particular, Acinus regulates the splicing of DFFA/ICAD transcript, a major regulator of DNA fragmentation. Globally, the genome-wide identification of RNA targets of Acinus revealed its role in splicing regulation as well as its involvement in other cellular pathways, including cell cycle progression. Altogether, this study uncovers new cellular functions of an RBP transiently associated with the EJC. PMID:27365209

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

    International Nuclear Information System (INIS)

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

  11. Genetic variations regulate alternative splicing in the 5' untranslated regions of the mouse glioma-associated oncogene 1, Gli1

    Directory of Open Access Journals (Sweden)

    Zaphiropoulos Peter G

    2010-04-01

    Full Text Available Abstract Background Alternative splicing is one of the key mechanisms that generate biological diversity. Even though alternative splicing also occurs in the 5' and 3' untranslated regions (UTRs of mRNAs, the understanding of the significance and the regulation of these variations is rather limited. Results We investigated 5' UTR mRNA variants of the mouse Gli1 oncogene, which is the terminal transcriptional effector of the Hedgehog (HH signaling pathway. In addition to identifying novel transcription start sites, we demonstrated that the expression ratio of the Gli1 splice variants in the 5' UTR is regulated by the genotype of the mouse strain analyzed. The GT allele, which contains the consensus intronic dinucleotides at the 5' splice site of intron 1B, favors exon 1B inclusion, while the GC allele, having a weaker 5' splice site sequence, promotes exon 1B skipping. Moreover, the alternative Gli1 5' UTRs had an impact on translational capacity, with the shorter and the exon 1B-skipped mRNA variants being most effective. Conclusions Our findings implicate novel, genome-based mechanisms as regulators of the terminal events in the mouse HH signaling cascade.

  12. Splicing-Sensitive DNA-Microarrays: Peculiarities and Applicationin Biomedical Research (Review

    Directory of Open Access Journals (Sweden)

    D.I. Knyazev

    2015-12-01

    Full Text Available Alternative splicing (АS provides a variety of protein and mature mRNA isoforms encoded by a single gene, and is the essential component of cell and tissue differentiation and functioning. DNA-microarrays are highly productive transcriptome research technique both at the level of total gene expression assessment and alternatively spliced mRNA isoforms exploration. The study of AS patterns requires thorough probe design to achieve appropriate accuracy of the analysis. There are two types of splicing-sensitive DNA-microarrays. The first type contain probes targeted to internal exonic sequences (exon bodies; the second type contain probes targeted to exon bodies and exon–exon and exon–intron junctions. So, the first section focused on probe sequence design, general features of splicing-sensitive DNA-microarrays and their main advantages and limitations. The results of AS research obtained using DNA-microarrays have been reviewed in special section. In particular, DNA-microarrays were used to reveal a number pre-mRNA processing and splicing mechanisms, to investigate AS patterns associated with cancer, cell and tissue differentiation. Splicing machinery regulation was demonstrated to be an essential step during carcinogenesis and differentiation. The examples of application of splicing-sensitive DNA-microarrays for diagnostic markers discovering and pathology mechanism elucidation were also reviewed. Investigations of AS role in pluripotency, stem cell commitment, immune and infected cells functioning during immune response are the promising future directions. Splicing-sensitive DNA-microarrays are relatively inexpensive but powerful research tool that give reason to suppose their introduction in clinical practice within the next few years.

  13. Trans-splicing repair of mutant p53 suppresses the growth of hepatocellular carcinoma cells in vitro and in vivo

    OpenAIRE

    Xingxing He; Fang Liu; Jingjun Yan; Yunan Zhang; Junwei Yan; Haitao Shang; Qian Dou; Qiu Zhao; Yuhu Song

    2015-01-01

    Reactivation of wild-type p53 (wt-p53) function is an attractive therapeutic approach to p53-defective cancers. An ideal p53-based gene therapy should restore wt-p53 production and reduces mutant p53 transcripts simultaneously. In this study, we described an alternative strategy named as trans-splicing that repaired mutant p53 transcripts in hepatocellular carcinoma (HCC) cells. The plasmids which encoded a pre-trans-splicing molecule (PTM) targeting intron 6 of p53 were constructed and then ...

  14. UBL5 is essential for pre-mRNA splicing and sister chromatid cohesion in human cells

    DEFF Research Database (Denmark)

    Oka, Yasuyoshi; Varmark, Hanne; Vitting-Seerup, Kristoffer;

    2014-01-01

    UBL5 is an atypical ubiquitin-like protein, whose function in metazoans remains largely unexplored. We show that UBL5 is required for sister chromatid cohesion maintenance in human cells. UBL5 primarily associates with spliceosomal proteins, and UBL5 depletion decreases pre-mRNA splicing efficiency......, leading to globally enhanced intron retention. Defective sister chromatid cohesion is a general consequence of dysfunctional pre-mRNA splicing, resulting from the selective downregulation of the cohesion protection factor Sororin. As the UBL5 yeast orthologue, Hub1, also promotes spliceosome functions...

  15. Inheritance of the group I rDNA intron in Tetrahymena pigmentosa

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Simon, E M; Engberg, J

    1992-01-01

    - strains looking for a strong polarity in the inheritance of the intron (intron homing). Based on the genetic analysis we find that the intron in T. pigmentosa is inherited as a neutral character and that intron+ and intron- alleles segregate in a Mendelian fashion with no sign of intron homing. In an...

  16. Contribution of bioinformatics predictions and functional splicing assays to the interpretation of unclassified variants of the BRCA genes

    Science.gov (United States)

    Théry, Jean Christophe; Krieger, Sophie; Gaildrat, Pascaline; Révillion, Françoise; Buisine, Marie-Pierre; Killian, Audrey; Duponchel, Christiane; Rousselin, Antoine; Vaur, Dominique; Peyrat, Jean-Philippe; Berthet, Pascaline; Frébourg, Thierry; Martins, Alexandra; Hardouin, Agnès; Tosi, Mario

    2011-01-01

    A large fraction of sequence variants of unknown significance (VUS) of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 may induce splicing defects. We analyzed 53 VUSs of BRCA1 or BRCA2, detected in consecutive molecular screenings, by using five splicing prediction programs, and we classified them into two groups according to the strength of the predictions. In parallel, we tested them by using functional splicing assays. A total of 10 VUSs were predicted by two or more programs to induce a significant reduction of splice site strength or activation of cryptic splice sites or generation of new splice sites. Minigene-based splicing assays confirmed four of these predictions. Five additional VUSs, all at internal exon positions, were not predicted to induce alterations of splice sites, but revealed variable levels of exon skipping, most likely induced by the modification of exonic splicing regulatory elements. We provide new data in favor of the pathogenic nature of the variants BRCA1 c.212+3A>G and BRCA1 c.5194−12G>A, which induced aberrant out-of-frame mRNA forms. Moreover, the novel variant BRCA2 c.7977−7C>G induced in frame inclusion of 6 nt from the 3′ end of intron 17. The novel variants BRCA2 c.520C>T and BRCA2 c.7992T>A induced incomplete skipping of exons 7 and 18, respectively. This work highlights the contribution of splicing minigene assays to the assessment of pathogenicity, not only when patient RNA is not available, but also as a tool to improve the accuracy of bioinformatics predictions. PMID:21673748

  17. MapSplice: Accurate mapping of RNA-seq reads for splice junction discovery

    OpenAIRE

    Kai WANG; Singh, Darshan; Zeng, Zheng; Coleman, Stephen J.; Huang, Yan; Savich, Gleb L.; He, Xiaping; Mieczkowski, Piotr; Grimm, Sara A; Perou, Charles M; MacLeod, James N; Chiang, Derek Y.; Prins, Jan F.; Liu, Jinze

    2010-01-01

    The accurate mapping of reads that span splice junctions is a critical component of all analytic techniques that work with RNA-seq data. We introduce a second generation splice detection algorithm, MapSplice, whose focus is high sensitivity and specificity in the detection of splices as well as CPU and memory efficiency. MapSplice can be applied to both short (

  18. A computational approach for prediction of donor splice sites with improved accuracy.

    Science.gov (United States)

    Meher, Prabina Kumar; Sahu, Tanmaya Kumar; Rao, A R; Wahi, S D

    2016-09-01

    Identification of splice sites is important due to their key role in predicting the exon-intron structure of protein coding genes. Though several approaches have been developed for the prediction of splice sites, further improvement in the prediction accuracy will help predict gene structure more accurately. This paper presents a computational approach for prediction of donor splice sites with higher accuracy. In this approach, true and false splice sites were first encoded into numeric vectors and then used as input in artificial neural network (ANN), support vector machine (SVM) and random forest (RF) for prediction. ANN and SVM were found to perform equally and better than RF, while tested on HS3D and NN269 datasets. Further, the performance of ANN, SVM and RF were analyzed by using an independent test set of 50 genes and found that the prediction accuracy of ANN was higher than that of SVM and RF. All the predictors achieved higher accuracy while compared with the existing methods like NNsplice, MEM, MDD, WMM, MM1, FSPLICE, GeneID and ASSP, using the independent test set. We have also developed an online prediction server (PreDOSS) available at http://cabgrid.res.in:8080/predoss, for prediction of donor splice sites using the proposed approach. PMID:27302911

  19. 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.

  20. A View of Pre-mRNA Splicing from RNase R Resistant RNAs

    Directory of Open Access Journals (Sweden)

    Hitoshi Suzuki

    2014-05-01

    Full Text Available During pre-mRNA splicing, exons in the primary transcript are precisely connected to generate an mRNA. Intron lariat RNAs are formed as by-products of this process. In addition, some exonic circular RNAs (circRNAs may also result from exon skipping as by-products. Lariat RNAs and circRNAs are both RNase R resistant RNAs. RNase R is a strong 3' to 5' exoribonuclease, which efficiently degrades linear RNAs, such as mRNAs and rRNAs; therefore, the circular parts of lariat RNAs and the circRNAs can be segregated from eukaryotic total RNAs by their RNase R resistance. Thus, RNase R resistant RNAs could provide unexplored splicing information not available from mRNAs. Analyses of these RNAs identified repeating splicing phenomena, such as re-splicing of mature mRNAs and nested splicing. Moreover, circRNA might function as microRNA sponges. There is an enormous variety of endogenous circRNAs, which are generally synthesized in cells and tissues.

  1. The Dengue Virus NS5 Protein Intrudes in the Cellular Spliceosome and Modulates Splicing.

    Science.gov (United States)

    De Maio, Federico A; Risso, Guillermo; Iglesias, Nestor G; Shah, Priya; Pozzi, Berta; Gebhard, Leopoldo G; Mammi, Pablo; Mancini, Estefania; Yanovsky, Marcelo J; Andino, Raul; Krogan, Nevan; Srebrow, Anabella; Gamarnik, Andrea V

    2016-08-01

    Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication. PMID:27575636

  2. Engineering splicing factors with designed specificities

    OpenAIRE

    Wang, Yang; Cheong, Cheom-Gil; Hall, Traci M Tanaka; Wang, Zefeng

    2009-01-01

    Alternative splicing is generally regulated by trans-acting factors that specifically bind pre-mRNA to activate or inhibit the splicing reaction. This regulation is critical for normal gene expression, and dysregulation of splicing is closely associated with human diseases. Here we engineer artificial splicing factors by combining sequence-specific RNA-binding domains of human Pumilio1 with functional domains that regulate splicing. We applied these factors to modulate different types of alte...

  3. Structure of the human laminin {gamma}2 chain gene (LAMC2): Alternative splicing with different tissue distribution of two transcripts

    Energy Technology Data Exchange (ETDEWEB)

    Airenne, T.; Haakana, H.; Kallunki, T. [Univ. of Oulu (Finland)] [and others

    1996-02-15

    This article discusses the exon-intron structure and tissue distribution of the laminin {gamma}2 chain (LAMC2) gene, which is mutated in some cases of junctional epidermolysis bullosa. The article also discusses the transcription and splicing of this gene, which result in alternative uses of the last two exons of the gene. The different tissue distributions of the transcripts indicate different functions for the gene in vivo. 36 refs., 8 figs., 3 tabs.

  4. Tissue-specific splicing mutation in acute intermittent porphyria

    International Nuclear Information System (INIS)

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

  5. Tissue-specific splicing mutation in acute intermittent porphyria

    Energy Technology Data Exchange (ETDEWEB)

    Grandchamp, B.; Picat, C. (Laboratoire de Genetique Moleculaire, Paris (France)); Mignotte, V.; Romeo, P.H.; Goossens, M. (Institut National de la Sante et de la Recherche Medicale, Creteil (France)); Wilson, J.H.P.; Sandkuyl, L. (Erasmus Univ., Rotterdam (Netherlands)); Te Velde, K. (Saint Geertruiden Hospital, Deventer (Netherlands)); Nordmann, Y. (Hopital Louis Mourier, Colombes (France))

    1989-01-01

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

  6. Integrating many co-splicing networks to reconstruct splicing regulatory modules

    OpenAIRE

    Dai Chao; Li Wenyuan; Liu Juan; Zhou Xianghong

    2012-01-01

    Abstract Background Alternative splicing is a ubiquitous gene regulatory mechanism that dramatically increases the complexity of the proteome. However, the mechanism for regulating alternative splicing is poorly understood, and study of coordinated splicing regulation has been limited to individual cases. To study genome-wide splicing regulation, we integrate many human RNA-seq datasets to identify splicing module, which we define as a set of cassette exons co-regulated by the same splicing f...

  7. Alternative Splicing Regulation During C. elegans Development: Splicing Factors as Regulated Targets

    OpenAIRE

    Sergio Barberan-Soler; Zahler, Alan M.

    2008-01-01

    Alternative splicing generates protein diversity and allows for post-transcriptional gene regulation. Estimates suggest that 10% of the genes in Caenorhabditis elegans undergo alternative splicing. We constructed a splicing-sensitive microarray to detect alternative splicing for 352 cassette exons and tested for changes in alternative splicing of these genes during development. We found that the microarray data predicted that 62/352 (approximately 18%) of the alternative splicing events studi...

  8. Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.

    Science.gov (United States)

    Zhang, Li; Tran, Ngoc-Tung; Su, Hairui; Wang, Rui; Lu, Yuheng; Tang, Haiping; Aoyagi, Sayura; Guo, Ailan; Khodadadi-Jamayran, Alireza; Zhou, Dewang; Qian, Kun; Hricik, Todd; Côté, Jocelyn; Han, Xiaosi; Zhou, Wenping; Laha, Suparna; Abdel-Wahab, Omar; Levine, Ross L; Raffel, Glen; Liu, Yanyan; Chen, Dongquan; Li, Haitao; Townes, Tim; Wang, Hengbin; Deng, Haiteng; Zheng, Y George; Leslie, Christina; Luo, Minkui; Zhao, Xinyang

    2015-01-01

    RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia. PMID:26575292

  9. ZmbZIP60 mRNA is spliced in maize in response to ER stress

    Directory of Open Access Journals (Sweden)

    Li Yanjie

    2012-03-01

    Full Text Available Abstract Background Adverse environmental conditions produce ER stress and elicit the unfolded protein response (UPR in plants. Plants are reported to have two "arms" of the ER stress signaling pathway-one arm involving membrane-bound transcription factors and the other involving a membrane-associated RNA splicing factor, IRE1. IRE1 in yeast to mammals recognizes a conserved twin loop structure in the target RNA. Results A segment of the mRNA encoding ZmbZIP60 in maize can be folded into a twin loop structure, and in response to ER stress this mRNA is spliced, excising a 20b intron. Splicing converts the predicted protein from a membrane-associated transcription factor to one that is targeted to the nucleus. Splicing of ZmbZIP60 can be elicited in maize seedlings by ER stress agents such as dithiothreitol (DTT or tunicamycin (TM or by heat treatment. Younger, rather than older seedlings display a more robust splicing response as do younger parts of leaf, along a developmental gradient in a leaf. The molecular signature of an ER stress response in plants includes the upregulation of Binding Protein (BIP genes. Maize has numerous BIP-like genes, and ER stress was found to upregulate one of these, ZmBIPb. Conclusions The splicing of ZmbZIP60 mRNA is an indicator of ER stress in maize seedlings resulting from adverse environmental conditions such as heat stress. ZmbZIP60 mRNA splicing in maize leads predictively to the formation of active bZIP transcription factor targeted to the nucleus to upregulate stress response genes. Among the genes upregulated by ER stress in maize is one of 22 BIP-like genes, ZmBIPb.

  10. Spliceosomal intron size expansion in domesticated grapevine (Vitis vinifera

    Directory of Open Access Journals (Sweden)

    Goertzen Leslie R

    2011-03-01

    Full Text Available Abstract Background Spliceosomal introns are important components of eukaryotic genes as their structure, sizes and contents reflect the architecture of gene and genomes. Intron size, determined by both neutral evolution, repetitive elements activities and potential functional constraints, varies significantly in eukaryotes, suggesting unique dynamics and evolution in different lineages of eukaryotic organisms. However, the evolution of intron size, is rarely studied. To investigate intron size dynamics in flowering plants, in particular domesticated grapevines, a survey of intron size and content in wine grape (Vitis vinifera Pinot Noir genes was conducted by assembling and mapping the transcriptome of V. vinifera genes from ESTs to characterize and analyze spliceosomal introns. Results Uncommonly large size of spliceosomal intron was observed in V. vinifera genome, otherwise inconsistent with overall genome size dynamics when comparing Arabidopsis, Populus and Vitis. In domesticated grapevine, intron size is generally not related to gene function. The composition of enlarged introns in grapevines indicated extensive transposable element (TE activity within intronic regions. TEs comprise about 80% of the expanded intron space and in particular, recent LTR retrotransposon insertions are enriched in these intronic regions, suggesting an intron size expansion in the lineage leading to domesticated grapevine, instead of size contractions in Arabidopsis and Populus. Comparative analysis of selected intronic regions in V. vinifera cultivars and wild grapevine species revealed that accelerated TE activity was associated with grapevine domestication, and in some cases with the development of specific cultivars. Conclusions In this study, we showed intron size expansion driven by TE activities in domesticated grapevines, likely a result of long-term vegetative propagation and intensive human care, which simultaneously promote TE proliferation and

  11. Patterns of intron gain and conservation in eukaryotic genes

    Directory of Open Access Journals (Sweden)

    Wolf Yuri I

    2007-10-01

    Full Text Available Abstract Background: The presence of introns in protein-coding genes is a universal feature of eukaryotic genome organization, and the genes of multicellular eukaryotes, typically, contain multiple introns, a substantial fraction of which share position in distant taxa, such as plants and animals. Depending on the methods and data sets used, researchers have reached opposite conclusions on the causes of the high fraction of shared introns in orthologous genes from distant eukaryotes. Some studies conclude that shared intron positions reflect, almost entirely, a remarkable evolutionary conservation, whereas others attribute it to parallel gain of introns. To resolve these contradictions, it is crucial to analyze the evolution of introns by using a model that minimally relies on arbitrary assumptions. Results: We developed a probabilistic model of evolution that allows for variability of intron gain and loss rates over branches of the phylogenetic tree, individual genes, and individual sites. Applying this model to an extended set of conserved eukaryotic genes, we find that parallel gain, on average, accounts for only ~8% of the shared intron positions. However, the distribution of parallel gains over the phylogenetic tree of eukaryotes is highly non-uniform. There are, practically, no parallel gains in closely related lineages, whereas for distant lineages, such as animals and plants, parallel gains appear to contribute up to 20% of the shared intron positions. In accord with these findings, we estimated that ancestral introns have a high probability to be retained in extant genomes, and conversely, that a substantial fraction of extant introns have retained their positions since the early stages of eukaryotic evolution. In addition, the density of sites that are available for intron insertion is estimated to be, approximately, one in seven basepairs. Conclusion: We obtained robust estimates of the contribution of parallel gain to the observed

  12. Whole exome sequencing in congenital pain insensitivity identifies a novel causative intronic NTRK1-mutation due to uniparental disomy.

    Science.gov (United States)

    Kurth, Ingo; Baumgartner, Manuela; Schabhüttl, Maria; Tomni, Cecilia; Windhager, Reinhard; Strom, Tim M; Wieland, Thomas; Gremel, Kurt; Auer-Grumbach, Michaela

    2016-09-01

    Congenital insensitivity to pain and anhidrosis (CIPA), also known as hereditary sensory and autonomic neuropathy type IV (HSAN IV), is characterized by recurrent episodes of unexplained high fever, loss of pain perception and temperature sensation, absent sweating, repeated traumatic and thermal injuries, and mild mental retardation. After exclusion of obviously pathogenic mutations in NTRK1, the most common cause of CIPA, whole exome sequencing (WES) was carried out in a CIPA patient with unrelated parents. No mutations in known HSAN genes were identified. However, filtering for genes carrying two rare sequence variations detected 13 homozygous single nucleotide variants (SNV), all being located on chromosome 1. Further analysis strongly suggested that this finding might be best explained by uniparental disomy of chromosome 1. Because NTRK1 is also located on chromosome 1, we re-evaluated WES data and detected a novel intronic sequence variation at position c.2188-12 C>A, homozygously because of uniparental disomy. Subsequent analysis of NTRK1 transcripts in peripheral blood cells of the patient revealed an influence of the variant on mRNA splicing. The C>A transversion generated a novel splice-site, which led to the incorporation of 10 intronic bases into the NTRK1 mRNA and consequently to a non-functional gene product. © 2016 Wiley Periodicals, Inc. PMID:27184211

  13. A novel splice variant in the N-propeptide of COL5A1 causes an EDS phenotype with severe kyphoscoliosis and eye involvement.

    Directory of Open Access Journals (Sweden)

    Sofie Symoens

    Full Text Available BACKGROUND: The Ehlers-Danlos Syndrome (EDS is a heritable connective tissue disorder characterized by hyperextensible skin, joint hypermobility and soft tissue fragility. The classic subtype of EDS is caused by mutations in one of the type V collagen genes (COL5A1 and COL5A2. Most mutations affect the type V collagen helical domain and lead to a diminished or structurally abnormal type V collagen protein. Remarkably, only two mutations were reported to affect the extended, highly conserved N-propeptide domain, which plays an important role in the regulation of the heterotypic collagen fibril diameter. We identified a novel COL5A1 N-propeptide mutation, resulting in an unusual but severe classic EDS phenotype and a remarkable splicing outcome. METHODOLOGY/PRINCIPAL FINDINGS: We identified a novel COL5A1 N-propeptide acceptor-splice site mutation (IVS6-2A>G, NM_000093.3_c.925-2A>G in a patient with cutaneous features of EDS, severe progressive scoliosis and eye involvement. Two mutant transcripts were identified, one with an exon 7 skip and one in which exon 7 and the upstream exon 6 are deleted. Both transcripts are expressed and secreted into the extracellular matrix, where they can participate in and perturb collagen fibrillogenesis, as illustrated by the presence of dermal collagen cauliflowers. Determination of the order of intron removal and computational analysis showed that simultaneous skipping of exons 6 and 7 is due to the combined effect of delayed splicing of intron 7, altered pre-mRNA secondary structure, low splice site strength and possibly disturbed binding of splicing factors. CONCLUSIONS/SIGNIFICANCE: We report a novel COL5A1 N-propeptide acceptor-splice site mutation in intron 6, which not only affects splicing of the adjacent exon 7, but also causes a splicing error of the upstream exon 6. Our findings add further insights into the COL5A1 splicing order and show for the first time that a single COL5A1 acceptor-splice site

  14. Unproductive alternative splicing and nonsense mRNAs: A widespread phenomenon among plant circadian clock genes

    Directory of Open Access Journals (Sweden)

    Filichkin Sergei A

    2012-07-01

    Full Text Available Abstract Background Recent mapping of eukaryotic transcriptomes and spliceomes using massively parallel RNA sequencing (RNA-seq has revealed that the extent of alternative splicing has been considerably underestimated. Evidence also suggests that many pre-mRNAs undergo unproductive alternative splicing resulting in incorporation of in-frame premature termination codons (PTCs. The destinies and potential functions of the PTC-harboring mRNAs remain poorly understood. Unproductive alternative splicing in circadian clock genes presents a special case study because the daily oscillations of protein expression levels require rapid and steep adjustments in mRNA levels. Results We conducted a systematic survey of alternative splicing of plant circadian clock genes using RNA-seq and found that many Arabidopsis thaliana circadian clock-associated genes are alternatively spliced. Results were confirmed using reverse transcription polymerase chain reaction (RT-PCR, quantitative RT-PCR (qRT-PCR, and/or Sanger sequencing. Intron retention events were frequently observed in mRNAs of the CCA1/LHY-like subfamily of MYB transcription factors. In contrast, the REVEILLE2 (RVE2 transcript was alternatively spliced via inclusion of a "poison cassette exon" (PCE. The PCE type events introducing in-frame PTCs are conserved in some mammalian and plant serine/arginine-rich splicing factors. For some circadian genes such as CCA1 the ratio of the productive isoform (i.e., a representative splice variant encoding the full-length protein to its PTC counterpart shifted sharply under specific environmental stress conditions. Conclusions Our results demonstrate that unproductive alternative splicing is a widespread phenomenon among plant circadian clock genes that frequently generates mRNA isoforms harboring in-frame PTCs. Because LHY and CCA1 are core components of the plant central circadian oscillator, the conservation of alternatively spliced variants between CCA1 and LHY

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

    Science.gov (United States)

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

    2005-10-01

    trans-Splicing, in which a spliced-leader (SL) RNA is appended to the most 5' exon of independently transcribed pre-mRNAs, has been described in a wide range of eukaryotes, from protozoans to chordates. Here we describe trans-splicing in the freshwater planarian Schmidtea mediterranea, a free-living member of the phylum Platyhelminthes. Analysis of an expressed sequence tag (EST) collection from this organism showed that over 300 transcripts shared one of two approximately 35-base sequences (Smed SL-1 and SL-2) at their 5' ends. Examination of genomic sequences encoding representatives of these transcripts revealed that these shared sequences were transcribed elsewhere in the genome. RNA blot analysis, 5' and 3' rapid amplification of cDNA ends, as well as genomic sequence data showed that 42-nt SL sequences were derived from small RNAs of approximately 110 nt. Similar sequences were also found at the 5' ends of ESTs from the planarian Dugesia japonica. trans-Splicing has already been described in numerous representatives of the phylum Platyhelminthes (trematodes, cestodes, and polyclads); its presence in two representatives of the triclads supports the hypothesis that this mode of RNA processing is ancestral within this group. The upcoming complete genome sequence of S. mediterranea, combined with this animal's experimental accessibility and susceptibility to RNAi, provide another model organism in which to study the function of the still-enigmatic trans-splicing. PMID:15972844

  16. Intronic polymorphisms of cytochromes P450

    Directory of Open Access Journals (Sweden)

    Ingelman-Sundberg Magnus

    2010-08-01

    Full Text Available Abstract The cytochrome P450 enzymes active in drug metabolism are highly polymorphic. Most allelic variants have been described for enzymes encoded by the cytochrome P450 family 2 (CYP2 gene family, which has 252 different alleles. The intronic polymorphisms in the cytochrome P450 genes account for only a small number of the important variant alleles; however, the most important ones are CYP2D6*4 and CYP2D6*41, which cause abolished and reduced CYP2D6 activity, respectively, and CYP3A5*3 and CYP3A5*5, common in Caucasian populations, which cause almost null activity. Their discoveries have been based on phenotypic alterations within individuals in a population, and their identification has, in several cases, been difficult and taken a long time. In light of the next-generation sequencing projects, it is anticipated that further alleles with intronic mutations will be identified that can explain the hitherto unidentified genetic basis of inter-individual differences in cytochrome P450-mediated drug and steroid metabolism.

  17. Co-option of the piRNA pathway for germline-specific alternative splicing of C. elegans TOR.

    Science.gov (United States)

    Barberán-Soler, Sergio; Fontrodona, Laura; Ribó, Anna; Lamm, Ayelet T; Iannone, Camilla; Cerón, Julián; Lehner, Ben; Valcárcel, Juan

    2014-09-25

    Many eukaryotic genes contain embedded antisense transcripts and repetitive sequences of unknown function. We report that male germline-specific expression of an antisense transcript contained in an intron of C. elegans Target of Rapamycin (TOR, let-363) is associated with (1) accumulation of endo-small interfering RNAs (siRNAs) against an embedded Helitron transposon and (2) activation of an alternative 3' splice site of TOR. The germline-specific Argonaute proteins PRG-1 and CSR-1, which participate in self/nonself RNA recognition, antagonistically regulate the generation of these endo-siRNAs, TOR mRNA levels, and 3' splice-site selection. Supply of exogenous double-stranded RNA against the region of sense/antisense overlap reverses changes in TOR expression and splicing and suppresses the progressive multigenerational sterility phenotype of prg-1 mutants. We propose that recognition of a "nonself" intronic transposon by endo-siRNAs/the piRNA system provides physiological regulation of expression and alternative splicing of a host gene that, in turn, contributes to the maintenance of germline function across generations. PMID:25220461

  18. 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/.

  19. Splice Site, Frameshift and Chimeric GFAP Mutations in Alexander Disease

    Science.gov (United States)

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

    2012-01-01

    Alexander disease (AxD) is a usually fatal astrogliopathy primarily caused by mutations in the gene encoding GFAP, an intermediate filament protein expressed in astrocytes. We describe three patients with unique characteristics, and whose mutations have implications for AxD diagnosis and studies of intermediate filaments. Patient 1 is the first reported case with a non-coding mutation. The patient has a splice site change producing an in-frame deletion of exon 4 in about 10% of the transcripts. Patient 2 has an insertion and deletion at the extreme end of the coding region, resulting in a short frameshift. In addition, the mutation was found in buccal DNA but not in blood DNA, making this patient the first reported chimera. Patient 3 has a single base deletion near the C-terminal end of the protein, producing a short frameshift. These findings recommend inclusion of intronic splice site regions in genetic testing for AxD, indicate that alteration of only a small fraction of GFAP can produce disease, and provide caution against tagging intermediate filaments at their C-terminal end for cell biological investigations. PMID:22488673

  20. Cytoplasmic male sterility of tuber mustard is associated with the alternative spliced mitochondrial T gene transcripts

    Institute of Scientific and Technical Information of China (English)

    PEI Yanxi; CHEN Zhujun; CAO Jiashu; CHEN Xuejun; LIU Xiaohui

    2004-01-01

    Two transcripts of T gene, T1170 and T1243, were obtained from the mitochondrial cDNA of tuber mustard CMS line. T1243 was a transcript with an intron unspliced, which has the basic characteristics of type Ⅱ intron. The expressions of the two transcripts were analyzed by reverse transcription PCR (RT-PCR). The results showed that, at seedling stage, the expression of T gene was mainly in the form of T1170 but decreased with the development gradually, while the expression abundance of another transcript, T1243, increased gradually. The T1243 was prevalent at the profuse flowering stage. The expression pattern was confirmed by Northern blot analysis. These results suggested that the alternative spliced mitochondrial T gene transcripts were related to CMS of tuber mustard.

  1. Intron-less RNA injected into the nucleus of Xenopus oocytes accesses a regulated translation control pathway.

    Science.gov (United States)

    Braddock, M; Muckenthaler, M; White, M R; Thorburn, A M; Sommerville, J; Kingsman, A J; Kingsman, S M

    1994-12-11

    The translation of a capped, polyadenylated RNA after injection into the nucleus of Xenopus oocytes occurs only if the RNA contains an intron. A single point mutation in the splice donor site prevents translation. Intron-less RNA is exported efficiently to the cytoplasm and is held, undegraded, in a translationally inert state for several days. Translation can be activated by treating the oocytes with progesterone or by injecting antibodies that bind the FRGY2 class of messenger RNA binding proteins, p56 and p60, but these antibodies are only effective if delivered to the nucleus. Inhibitors of casein kinase II also activate translation whereas phosphatase inhibitors block progesterone-mediated activation of translation. These data suggest the presence of an RNA handling pathway in the nucleus of Xenopus oocytes which is regulated by casein kinase type II phosphorylation and which directs transcripts to be sequestered by p56/p60 or by closely related proteins. This pathway can be bypassed if the RNA contains an intron and it can be reversed by progesterone treatment. These data may have implications for understanding translational control during early development. PMID:7816614

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

    International Nuclear Information System (INIS)

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

  3. Post-transcriptional repair of a split heat shock protein 90 gene by mRNA trans-splicing.

    Science.gov (United States)

    Nageshan, Rishi Kumar; Roy, Nainita; Hehl, Adrian B; Tatu, Utpal

    2011-03-01

    Heat shock protein 90 participates in diverse biological processes ranging from protein folding, cell cycle, signal transduction and development to evolution in all eukaryotes. It is also critically involved in regulating growth of protozoa such as Dictyostelium discoideum, Leishmania donovani, Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma evansi. Selective inhibition of Hsp90 has also been explored as an intervention strategy against important human diseases such as cancer, malaria, or trypanosomiasis. Giardia lamblia, a simple protozoan parasite of humans and animals, is an important cause of diarrheal disease with significant morbidity and some mortality in tropical countries. Here we show that the G. lamblia cytosolic hsp90 (glhsp90) is split in two similar sized fragments located 777 kb apart on the same scaffold. Intrigued by this unique arrangement, which appears to be specific for the Giardiinae, we have investigated the biosynthesis of GlHsp90. We used genome sequencing to confirm the split nature of the giardial hsp90. However, a specific antibody raised against the peptide detected a product with a mass of about 80 kDa, suggesting a post-transcriptional rescue of the genomic defect. We show evidence for the joining of the two independent Hsp90 transcripts in-trans to one long mature mRNA presumably by RNA splicing. The splicing junction carries hallmarks of classical cis-spliced introns, suggesting that the regular cis-splicing machinery may be sufficient for repair of the open reading frame. A complementary 26-nt sequence in the "intron" regions adjacent to the splice sites may assist in positioning the two pre-mRNAs for processing. This is the first example of post-transcriptional rescue of a split gene by trans-splicing. PMID:21209094

  4. Recognizing the 35th anniversary of the proposal that snRNPs are involved in splicing

    OpenAIRE

    Mount, Stephen M; Wolin, Sandra L.

    2015-01-01

    Thirty-five years ago, as young graduate students, we had the pleasure and privilege of being in Joan Steitz’s laboratory at a pivotal point in the history of RNA molecular biology. Introns had recently been discovered in the laboratories of Philip Sharp and Richard Roberts, but the machinery for removing them from mRNA precursors was entirely unknown. This Retrospective describes our hypothesis that recently discovered snRNPs functioned in pre-mRNA splicing. The proposal was proven correct, ...

  5. Genetic algorithms with exons and introns for the satisfiability problem

    OpenAIRE

    Popov, V.

    2013-01-01

    In this paper we propose a new model of genetic algorithms. This model uses notions of exons and introns. We consider the satisfiability problem as a testbed for a genetic algorithm with exons and introns. © 2013 Lhachmi El Badri et al.

  6. EASI—enrichment of alternatively spliced isoforms

    OpenAIRE

    Julian P Venables; Burn, John

    2006-01-01

    Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This ...

  7. Mechano-Regulation of Alternative Splicing

    OpenAIRE

    Liu, Huan; Tang, Liling

    2013-01-01

    Alternative splicing contributes to the complexity of proteome by producing multiple mRNAs from a single gene. Affymetrix exon arrays and experiments in vivo or in vitro demonstrated that alternative splicing was regulated by mechanical stress. Expression of mechano-growth factor (MGF) which is the splicing isoform of insulin-like growth factor 1(IGF-1) and vascular endothelial growth factor (VEGF) splicing variants such as VEGF121, VEGF165, VEGF206, VEGF189, VEGF165 and VEGF145 are regulated...

  8. ASD: a bioinformatics resource on alternative splicing

    OpenAIRE

    Stamm, Stefan; Riethoven, Jean-Jack; Le Texier, Vincent; Gopalakrishnan, Chellappa; Kumanduri, Vasudev; Tang, Yesheng; Barbosa-Morais, Nuno L.; Thanaraj, Thangavel Alphonse

    2005-01-01

    Alternative splicing is an important regulatory mechanism of mammalian gene expression. The alternative splicing database (ASD) consortium is systematically collecting and annotating data on alternative splicing. We present the continuation and upgrade of the ASD [T. A. Thanaraj, S. Stamm, F. Clark, J. J. Riethoven, V. Le Texier, J. Muilu (2004) Nucleic Acids Res. 32, D64–D69] that consists of computationally and manually generated data. Its largest parts are AltSplice, a value-added database...

  9. Targeting RNA Splicing for Disease Therapy

    OpenAIRE

    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 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 splicin...

  10. Evolution of alternative splicing after gene duplication

    OpenAIRE

    Su, Zhixi; Wang, Jianmin; Yu, Jun; Huang, Xiaoqiu; Gu, Xun

    2006-01-01

    Alternative splicing and gene duplication are two major sources of proteomic function diversity. Here, we study the evolutionary trend of alternative splicing after gene duplication by analyzing the alternative splicing differences between duplicate genes. We observed that duplicate genes have fewer alternative splice (AS) forms than single-copy genes, and that a negative correlation exists between the mean number of AS forms and the gene family size. Interestingly, we found that the loss of ...

  11. A biophysical model for identifying splicing regulatory elements and their interactions.

    Directory of Open Access Journals (Sweden)

    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.

  12. Mutant allele of rna14 in fission yeast affects pre-mRNA splicing

    Indian Academy of Sciences (India)

    SUDHANSHU YADAV; AMIT SONKAR; NAFEES AHAMAD; SHAKIL AHMED

    2016-06-01

    complex removes noncoding introns, while 3'end processing involves in cleavage and addition of poly(A) tails to the nascent transcript. Rna14 protein in budding yeast has been implicated in cleavage and polyadenylation of mRNA in the nucleus but their role in the pre-mRNA splicing has not been studied. Here, we report the isolation of a mutant allele of rna14 in fission yeast,Schizosaccharomyces pombe that exhibits reduction in protein level of Chk1 at the nonpermissive temperature, primarily due to the defects in posttranscriptional processing. Reverse transcriptase-polymerase chain reaction analysis reveals defective splicing of the chk1¹+transcript at the nonpermissive temperature. Apart from chk1¹+, the splicing of some other genes were also found to be defective at the nonpermissive temperature suggesting that Rna14 might be involved in pre-mRNA splicing. Subsequently, genetic interaction of Rna14 with prp1 and physical interactions with Prp28 suggest that the Rna14 might be part of a larger protein complex responsible for the pre-mRNA maturation.

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

    OpenAIRE

    Kang Ning; Damian Fermin

    2010-01-01

    Splicing event identification is one of the most important issues in the comprehensive analysis of transcription profile. Recent development of next-generation sequencing technology has generated an extensive profile of alternative splicing. However, while many of these splicing events are between exons that are relatively close on genome sequences, reads generated by RNA-Seq are not limited to alternative splicing between close exons but occur in virtually all splicing events. In this work, ...

  14. Mutation in the U2 snRNA influences exon interactions of U5 snRNA loop 1 during pre-mRNA splicing

    OpenAIRE

    McGrail, Joanne C.; Tatum, Elaine M; O'Keefe, Raymond T.

    2006-01-01

    The U2 and U6 snRNAs contribute to the catalysis of intron removal while U5 snRNA loop 1 holds the exons for ligation during pre-mRNA splicing. It is unclear how different exons are positioned precisely with U5 loop 1. Here, we investigate the role of U2 and U6 in positioning the exons with U5 loop 1. Reconstitution in vitro of spliceosomes with mutations in U2 allows U5–pre-mRNA interactions before the first step of splicing. However, insertion in U2 helix Ia disrupts U5–exon interactions wi...

  15. hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells

    OpenAIRE

    Chou, Min-Yuan; Rooke, Nanette; Turck, Christoph W.; Black, Douglas L.

    1999-01-01

    The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5′ splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfa...

  16. Genetic analysis of complement C1s deficiency associated with systemic lupus erythematosus highlights alternative splicing of normal C1s gene

    DEFF Research Database (Denmark)

    Armano, MT; Ferriani, VP; Florido, MP;

    2008-01-01

    ' fibroblasts when analyzed by confocal microscopy. We show that all four siblings are homozygous for a mutation at position 938 in exon 6 of the C1s cDNA that creates a premature stop codon. Our investigations led us to reveal the presence of previously uncharacterized splice variants of C1s mRNA transcripts...... in normal human cells. These variants are derived from the skipping of exon 3 and from the use of an alternative 3' splice site within intron 1 which increases the size of exon 2 by 87 nucleotides....

  17. The Pentatricopeptide Repeat Proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 Are Involved in the Splicing of the Multipartite nad5 Transcript Encoding a Subunit of Mitochondrial Complex I.

    Science.gov (United States)

    Colas des Francs-Small, Catherine; Falcon de Longevialle, Andéol; Li, Yunhai; Lowe, Elizabeth; Tanz, Sandra K; Smith, Caroline; Bevan, Michael W; Small, Ian

    2014-06-23

    Pentatricopeptide repeat proteins constitute a large family of RNA-binding proteins in higher plants (around 450 genes in Arabidopsis [Arabidopsis thaliana]), mostly targeted to chloroplasts and mitochondria. Many of them are involved in organelle posttranscriptional processes, in a very specific manner. Splicing is necessary to remove the group II introns, which interrupt the coding sequences of several genes encoding components of the mitochondrial respiratory chain. The nad5 gene is fragmented in five exons, belonging to three distinct transcription units. Its maturation requires two cis- and two trans-splicing events. These steps need to be performed in a very precise order to generate a functional transcript. Here, we characterize two pentatricopeptide repeat proteins, ORGANELLE TRANSCRIPT PROCESSING439 and TANG2, and show that they are involved in the removal of nad5 introns 2 and 3, respectively. To our knowledge, they are the first two specific nad5 splicing factors found in plants so far. PMID:24958715

  18. The Pentatricopeptide Repeat Proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 Are Involved in the Splicing of the Multipartite nad5 Transcript Encoding a Subunit of Mitochondrial Complex I1[W][OPEN

    Science.gov (United States)

    Colas des Francs-Small, Catherine; Falcon de Longevialle, Andéol; Li, Yunhai; Lowe, Elizabeth; Tanz, Sandra K.; Smith, Caroline; Bevan, Michael W.; Small, Ian

    2014-01-01

    Pentatricopeptide repeat proteins constitute a large family of RNA-binding proteins in higher plants (around 450 genes in Arabidopsis [Arabidopsis thaliana]), mostly targeted to chloroplasts and mitochondria. Many of them are involved in organelle posttranscriptional processes, in a very specific manner. Splicing is necessary to remove the group II introns, which interrupt the coding sequences of several genes encoding components of the mitochondrial respiratory chain. The nad5 gene is fragmented in five exons, belonging to three distinct transcription units. Its maturation requires two cis- and two trans-splicing events. These steps need to be performed in a very precise order to generate a functional transcript. Here, we characterize two pentatricopeptide repeat proteins, ORGANELLE TRANSCRIPT PROCESSING439 and TANG2, and show that they are involved in the removal of nad5 introns 2 and 3, respectively. To our knowledge, they are the first two specific nad5 splicing factors found in plants so far. PMID:24958715

  19. Conserved intron positions in FGFR genes reflect the modular structure of FGFR and reveal stepwise addition of domains to an already complex ancestral FGFR.

    Science.gov (United States)

    Rebscher, Nicole; Deichmann, Christina; Sudhop, Stefanie; Fritzenwanker, Jens Holger; Green, Stephen; Hassel, Monika

    2009-10-01

    We have analyzed the evolution of fibroblast growth factor receptor (FGFR) tyrosine kinase genes throughout a wide range of animal phyla. No evidence for an FGFR gene was found in Porifera, but we tentatively identified an FGFR gene in the placozoan Trichoplax adhaerens. The gene encodes a protein with three immunoglobulin-like domains, a single-pass transmembrane, and a split tyrosine kinase domain. By superimposing intron positions of 20 FGFR genes from Placozoa, Cnidaria, Protostomia, and Deuterostomia over the respective protein domain structure, we identified ten ancestral introns and three conserved intron groups. Our analysis shows (1) that the position of ancestral introns correlates to the modular structure of FGFRs, (2) that the acidic domain very likely evolved in the last common ancestor of triploblasts, (3) that splicing of IgIII was enabled by a triploblast-specific insertion, and (4) that IgI is subject to substantial loss or duplication particularly in quickly evolving genomes. Moreover, intron positions in the catalytic domain of FGFRs map to the borders of protein subdomains highly conserved in other serine/threonine kinases. Nevertheless, these introns were introduced in metazoan receptor tyrosine kinases exclusively. Our data support the view that protein evolution dating back to the Cambrian explosion took place in such a short time window that only subtle changes in the domain structure are detectable in extant representatives of animal phyla. We propose that the first multidomain FGFR originated in the last common ancestor of Placozoa, Cnidaria, and Bilateria. Additional domains were introduced mainly in the ancestor of triploblasts and in the Ecdysozoa. PMID:20016912

  20. Multiple splice defects in ABCA1 cause low HDL-C in a family with Hypoalphalipoproteinemia and premature coronary disease

    Directory of Open Access Journals (Sweden)

    Miller Michael

    2009-01-01

    Full Text Available Abstract Background Mutations at splice junctions causing exon skipping are uncommon compared to exonic mutations, and two intronic mutations causing an aberrant phenotype have rarely been reported. Despite the high number of functional ABCA1 mutations reported to date, splice variants have been reported infrequently. We screened DNA from a 41 year-old male with low HDL-C (12 mg/dL [0.31 mmol/L] and a family history of premature coronary heart disease (CHD using polymerase chain reaction single-strand conformation polymorphism (SSCP analysis. Methods Family members with low levels of HDL-C (n = 6 were screened by SSCP for mutations in ABCA1. Samples with altered SSCP patterns were sequenced directly using either an ABI 3700 or ABI3730Xl DNA Analyzer. To screen for splicing defects, cDNA was isolated from the proband's RNA and was sequenced as above. A series of minigenes were constructed to determine the contribution of normal and defective alleles. Results Two novel splice variants in ABCA1 were identified. The first mutation was a single base pair change (T->C in IVS 7, 6 bps downstream from the exon7/intron7 junction. Amplification of cDNA and allelic subcloning identified skipping of Exon 7 that results in the elimination of 59 amino acids from the first extracellular loop of the ABCA1 protein. The second mutation was a single base pair change (G->C at IVS 31 -1, at the intron/exon junction of exon 32. This mutation causes skipping of exon 32, resulting in 8 novel amino acids followed by a stop codon and a predicted protein size of 1496 AA, compared to normal (2261 AA. Bioinformatic studies predicted an impact on splicing as confirmed by in vitro assays of constitutive splicing. Conclusion In addition to carnitine-acylcarnitine translocase (CACT deficiency and Hermansky-Pudlak syndrome type 3, this represents only the third reported case in which 2 different splice mutations has resulted in an aberrant clinical phenotype.

  1. Multiple splice defects in ABCA1 cause low HDL-C in a family with Hypoalphalipoproteinemia and premature coronary disease

    Science.gov (United States)

    Rhyne, Jeffrey; Mantaring, Myrna M; Gardner, David F; Miller, Michael

    2009-01-01

    Background Mutations at splice junctions causing exon skipping are uncommon compared to exonic mutations, and two intronic mutations causing an aberrant phenotype have rarely been reported. Despite the high number of functional ABCA1 mutations reported to date, splice variants have been reported infrequently. We screened DNA from a 41 year-old male with low HDL-C (12 mg/dL [0.31 mmol/L]) and a family history of premature coronary heart disease (CHD) using polymerase chain reaction single-strand conformation polymorphism (SSCP) analysis. Methods Family members with low levels of HDL-C (n = 6) were screened by SSCP for mutations in ABCA1. Samples with altered SSCP patterns were sequenced directly using either an ABI 3700 or ABI3730Xl DNA Analyzer. To screen for splicing defects, cDNA was isolated from the proband's RNA and was sequenced as above. A series of minigenes were constructed to determine the contribution of normal and defective alleles. Results Two novel splice variants in ABCA1 were identified. The first mutation was a single base pair change (T->C) in IVS 7, 6 bps downstream from the exon7/intron7 junction. Amplification of cDNA and allelic subcloning identified skipping of Exon 7 that results in the elimination of 59 amino acids from the first extracellular loop of the ABCA1 protein. The second mutation was a single base pair change (G->C) at IVS 31 -1, at the intron/exon junction of exon 32. This mutation causes skipping of exon 32, resulting in 8 novel amino acids followed by a stop codon and a predicted protein size of 1496 AA, compared to normal (2261 AA). Bioinformatic studies predicted an impact on splicing as confirmed by in vitro assays of constitutive splicing. Conclusion In addition to carnitine-acylcarnitine translocase (CACT) deficiency and Hermansky-Pudlak syndrome type 3, this represents only the third reported case in which 2 different splice mutations has resulted in an aberrant clinical phenotype. PMID:19133158

  2. 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.

  3. A Splice Region Variant in LDLR Lowers Non-high Density Lipoprotein Cholesterol and Protects against Coronary Artery Disease.

    Directory of Open Access Journals (Sweden)

    Solveig Gretarsdottir

    2015-09-01

    Full Text Available Through high coverage whole-genome sequencing and imputation of the identified variants into a large fraction of the Icelandic population, we found four independent signals in the low density lipoprotein receptor gene (LDLR that associate with levels of non-high density lipoprotein cholesterol (non-HDL-C and coronary artery disease (CAD. Two signals are novel with respect to association with non-HDL-C and are represented by non-coding low frequency variants (between 2-4% frequency, the splice region variant rs72658867-A in intron 14 and rs17248748-T in intron one. These two novel associations were replicated in three additional populations. Both variants lower non-HDL-C levels (rs72658867-A, non-HDL-C effect = -0.44 mmol/l, Padj = 1.1 × 10⁻⁸⁰ and rs17248748-T, non-HDL-C effect = -0.13 mmol/l, Padj = 1.3 × 10⁻¹² and confer protection against CAD (rs72658867-A, OR = 0.76 and Padj = 2.7 × 10⁻⁸ and rs17248748-T, OR = 0.92 and Padj = 0.022. The LDLR splice region variant, rs72658867-A, located at position +5 in intron 14 (NM_000527:c.2140+5G>A, causes retention of intron 14 during transcription and is expected to produce a truncated LDL receptor lacking domains essential for function of the receptor. About half of the transcripts generated from chromosomes carrying rs72658867-A are characterized by this retention of the intron. The same variant also increases LDLR mRNA expression, however, the wild type transcripts do not exceed levels in non-carriers. This demonstrates that sequence variants that disrupt the LDL receptor can lower non-HDL-C and protect against CAD.

  4. 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.

  5. Intron retention generates a novel isoform of the murine vitamin D receptor that acts in a dominant negative way on the vitamin D signaling pathway.

    OpenAIRE

    Ebihara, K; Masuhiro, Y; Kitamoto, T; Suzawa, M; Uematsu, Y; Yoshizawa, T; Ono, T.; Harada, H; Matsuda, K; Hasegawa, T; Masushige, S; S. Kato

    1996-01-01

    We identified and characterized a novel rat vitamin D receptor isoform (rVDR1), which retains intron 8 of the canonical VDR (rVDR0) during alternative splicing. In this isoform protein directed by the stop codon in this newly identified exon, a part of the ligand binding domain (86 amino acids) is truncated at the C-terminal end but contains 19 extra amino acids. The rVDR1 transcript was expressed at a level 1/15 to 1/20 of that of rVDR0 in the kidney and intestine in adult rats but not in em...

  6. Exon-level transcriptome profiling in murine breast cancer reveals splicing changes specific to tumors with different metastatic abilities.

    Directory of Open Access Journals (Sweden)

    Amandine Bemmo

    Full Text Available BACKGROUND: Breast cancer is the second most frequent type of cancer affecting women. We are increasingly aware that changes in mRNA splicing are associated with various characteristics of cancer. The most deadly aspect of cancer is metastasis, the process by which cancer spreads from the primary tumor to distant organs. However, little is known specifically about the involvement of alternative splicing in the formation of macroscopic metastases. Our study investigates transcript isoform changes that characterize tumors of different abilities to form growing metastases. METHODS AND FINDINGS: To identify alternative splicing events (ASEs that are associated with the fully metastatic phenotype in breast cancer, we used Affymetrix Exon Microarrays to profile mRNA isoform variations genome-wide in weakly metastatic (168FARN and 4T07 and highly metastatic (4T1 mammary carcinomas. Statistical analysis identified significant expression changes in 7606 out of 155,994 (4% exons and in 1725 out of 189,460 (1% intronic regions, which affect 2623 out of 16,654 (16% genes. These changes correspond to putative alternative isoforms-several of which are novel-that are differentially expressed between tumors of varying metastatic phenotypes. Gene pathway analysis showed that 1224 of genes expressing alternative isoforms were involved in cell growth, cell interactions, cell proliferation, cell migration and cell death and have been previously linked to cancers and genetic disorders. We chose ten predicted splice variants for RT-PCR validation, eight of which were successfully confirmed (MED24, MFI2, SRRT, CD44, CLK1 and HNRNPH1. These include three novel intron retentions in CD44, a gene in which isoform variations have been previously associated with the metastasis of several cancers. CONCLUSION: Our findings reveal that various genes are differently spliced and/or expressed in association with the metastatic phenotype of tumor cells. Identification of

  7. 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.

  8. Survivin 2α: a novel Survivin splice variant expressed in human malignancies

    Directory of Open Access Journals (Sweden)

    Honsey Laura E

    2005-03-01

    Full Text Available Abstract Background Survivin and its alternative splice forms are involved in critical cellular processes, including cell division and programmed cell death. Survivin is expressed in the majority of human cancers, but minimally in differentiated normal tissues. Expression levels correlate with tumor aggressiveness and resistance to therapy. Results In the present study, we identify and characterize a novel survivin isoform that we designate survivin 2α. Structurally, the transcript consists of 2 exons: exon 1 and exon 2, as well as a 3' 197 bp region of intron 2. Acquisition of a new in-frame stop codon within intron 2 results in an open reading frame of 225 nucleotides, predicting a truncated 74 amino acid protein. Survivin 2α is expressed at high levels in several malignant cell lines and primary tumors. Functional assays show that survivin 2α attenuates the anti-apoptotic activity of survivin. Subcellular localization and immunoprecipitation of survivin 2α suggests a physical interaction with survivin. Conclusion We characterized a novel survivin splice variant that we designated survivin 2α. We hypothesize that survivin 2α can alter the anti-apoptotic functions of survivin in malignant cells. Thus survivin 2α may be useful as a therapeutic tool in sensitizing chemoresistant tumor cells to chemotherapy.

  9. Ire1 Has Distinct Catalytic Mechanisms for XBP1/HAC1 Splicing and RIDD

    Directory of Open Access Journals (Sweden)

    Arvin B. Tam

    2014-11-01

    Full Text Available An evolutionarily conserved unfolded protein response (UPR component, IRE1, cleaves XBP1/HAC1 introns in order to generate spliced mRNAs that are translated into potent transcription factors. IRE1 also cleaves endoplasmic-reticulum-associated RNAs leading to their decay, an activity termed regulated IRE1-dependent decay (RIDD; however, the mechanism by which IRE1 differentiates intron cleavage from RIDD is not well understood. Using in vitro experiments, we found that IRE1 has two different modes of action: XBP1/HAC1 is cleaved by IRE1 subunits acting cooperatively within IRE1 oligomers, whereas a single subunit of IRE1 performs RIDD without cooperativity. Furthermore, these distinct activities can be separated by complementation of catalytically inactive IRE1 RNase and mutations at oligomerization interfaces. Using an IRE1 RNase inhibitor, STF-083010, selective inhibition of XBP1 splicing indicates that XBP1 promotes cell survival, whereas RIDD leads to cell death, revealing modulation of IRE1 activities as a drug-development strategy.

  10. Inteins, introns, and homing endonucleases: recent revelations about the life cycle of parasitic genetic elements

    Directory of Open Access Journals (Sweden)

    Hilario Elena

    2006-11-01

    Full Text Available Abstract Self splicing introns and inteins that rely on a homing endonuclease for propagation are parasitic genetic elements. Their life-cycle and evolutionary fate has been described through the homing cycle. According to this model the homing endonuclease is selected for function only during the spreading phase of the parasite. This phase ends when the parasitic element is fixed in the population. Upon fixation the homing endonuclease is no longer under selection, and its activity is lost through random processes. Recent analyses of these parasitic elements with functional homing endonucleases suggest that this model in its most simple form is not always applicable. Apparently, functioning homing endonuclease can persist over long evolutionary times in populations and species that are thought to be asexual or nearly asexual. Here we review these recent findings and discuss their implications. Reasons for the long-term persistence of a functional homing endonuclease include: More recombination (sexual and as a result of gene transfer than previously assumed for these organisms; complex population structures that prevent the element from being fixed; a balance between active spreading of the homing endonuclease and a decrease in fitness caused by the parasite in the host organism; or a function of the homing endonuclease that increases the fitness of the host organism and results in purifying selection for the homing endonuclease activity, even after fixation in a local population. In the future, more detailed studies of the population dynamics of the activity and regulation of homing endonucleases are needed to decide between these possibilities, and to determine their relative contributions to the long term survival of parasitic genes within a population. Two outstanding publications on the amoeba Naegleria group I intron (Wikmark et al. BMC Evol Biol 2006, 6:39 and the PRP8 inteins in ascomycetes (Butler et al.BMC Evol Biol 2006, 6:42 provide

  11. SpliceProt: a protein sequence repository of predicted human splice variants.

    Science.gov (United States)

    Tavares, Raphael; de Miranda Scherer, Nicole; Pauletti, Bianca Alves; Araújo, Elói; Folador, Edson Luiz; Espindola, Gabriel; Ferreira, Carlos Gil; Paes Leme, Adriana Franco; de Oliveira, Paulo Sergio Lopes; Passetti, Fabio

    2014-02-01

    The mechanism of alternative splicing in the transcriptome may increase the proteome diversity in eukaryotes. In proteomics, several studies aim to use protein sequence repositories to annotate MS experiments or to detect differentially expressed proteins. However, the available protein sequence repositories are not designed to fully detect protein isoforms derived from mRNA splice variants. To foster knowledge for the field, here we introduce SpliceProt, a new protein sequence repository of transcriptome experimental data used to investigate for putative splice variants in human proteomes. Current version of SpliceProt contains 159 719 non-redundant putative polypeptide sequences. The assessment of the potential of SpliceProt in detecting new protein isoforms resulting from alternative splicing was performed by using publicly available proteomics data. We detected 173 peptides hypothetically derived from splice variants, which 54 of them are not present in UniprotKB/TrEMBL sequence repository. In comparison to other protein sequence repositories, SpliceProt contains a greater number of unique peptides and is able to detect more splice variants. Therefore, SpliceProt provides a solution for the annotation of proteomics experiments regarding splice isofoms. The repository files containing the translated sequences of the predicted splice variants and a visualization tool are freely available at http://lbbc.inca.gov.br/spliceprot. PMID:24273012

  12. Post-harvest regulated gene expression and splicing efficiency in storage roots of sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Rotthues, Alexander; Kappler, Jeannette; Lichtfuss, Anna; Kloos, Dorothee U; Stahl, Dietmar J; Hehl, Reinhard

    2008-05-01

    Sixteen post-harvest upregulated genes from sugar beet comprising five novel sequences were isolated by subtractive cloning. Transcription profiles covering a period of up to 49 days after harvest under controlled storage conditions and in field clamps are reported. Post-harvest induced genes are involved in wound response, pathogen defense, dehydration stress, and detoxification of reactive oxygen species. An early induction of a cationic peroxidase indicates a response to post-harvest damage. Wound response reactions may also involve genes required for cell division such as a regulator of chromatin condensation and a precursor of the growth stimulating peptide phytohormone phytosulfokine-alpha. Surprisingly, also three putative non-protein coding genes were isolated. Two of these genes show intron specific and storage temperature dependent splicing of a precursor mRNA. The temperature dependent splicing of an intron containing sugar beet mRNA is also maintained in transgenic Arabidopsis thaliana. The storage induced genes are integrated into a model that proposes the response to several post-harvest stress conditions. Temperature regulated splicing may be a mechanism to sense seasonal temperature changes. PMID:18324413

  13. Deletions in cox2 mRNA result in loss of splicing and RNA editing and gain of novel RNA editing sites.

    Directory of Open Access Journals (Sweden)

    Stefanie Grüttner

    Full Text Available As previously demonstrated, the maize cox2 RNA is fully edited in cauliflower mitochondria. Use of constructs with a deleted cox2 intron, however, led to a loss of RNA editing at almost all editing sites, with only a few sites still partially edited. Likewise, one deletion in exon 1 and three in exon 2 abolish RNA editing at all cox2 sites analyzed. Furthermore, intron splicing is abolished using these deletions. Mutation of a cytosine residue, which is normally edited and localized directly adjacent to the intron, to thymidine did not result in restoration of splicing, indicating that the loss of splicing was not due to loss of RNA editing. One deletion in exon 2 did not lead to loss of splicing. Instead, most editing sites were found to be edited, only three were not edited. Unexpectedly, we observed additional RNA editing events at new sites. Thus it appears that deletions in the cox2 RNA sequence can have a strong effect on RNA processing, leading to loss of splicing, loss of editing at all sites, or even to a gain of new editing sites. As these effects are not limited to the vicinity of the respective deletions, but appear to be widespread or even affect all editing sites, they may not be explained by the loss of PPR binding sites. Instead, it appears that several parts of the cox2 transcript are required for proper RNA processing. This indicates the roles of the RNA sequence and structural elements in the recognition of the editing sites.

  14. Exon coconversion biases accompanying intron homing: battle of the nucleases.

    Science.gov (United States)

    Mueller, J E; Smith, D; Belfort, M

    1996-09-01

    Intron homing in phage T4 occurs in the context of recombination-dependent replication, by virtue of intron-encoded endonucleolytic activity. After the td intron endonuclease I-TevI cleaves the intronless recipient 23 and 25 nucleotides upstream of the intron insertion site, exonucleolytic degradation is required for recombination to proceed. This resection process results in coconversion of exon sequences flanking the intron. In a genetic system designed to study coconversion of flanking markers, we demonstrate that although there is a bidirectional polarity gradient, coconversion can be highly asymmetric. Furthermore, we show that the coconversion of flanking markers favors exon I sequences, upstream of the I-TevI cleavage site. These data are consistent with the asymmetric features of the homing pathways that have been invoked for intron mobility in phage T4. Moreover, these results are in accord with the finding that once the td homing-site substrate is cleaved, I-TevI remains bound to the downstream cleavage product, protecting against exonucleolytic degradation, and thereby limiting the extent of coconversion into exon II. The results suggest that recombination events are influenced by a competition between the homing endonuclease and exonucleases for sequences downstream of the I-TevI cleavage site, thereby implying a role for the homing endonuclease in the repair process. PMID:8804310

  15. Unexpected role of the steroid-deficiency protein ecdysoneless in pre-mRNA splicing.

    Directory of Open Access Journals (Sweden)

    Ann-Katrin Claudius

    2014-04-01

    Full Text Available The steroid hormone ecdysone coordinates insect growth and development, directing the major postembryonic transition of forms, metamorphosis. The steroid-deficient ecdysoneless1 (ecd1 strain of Drosophila melanogaster has long served to assess the impact of ecdysone on gene regulation, morphogenesis, or reproduction. However, ecd also exerts cell-autonomous effects independently of the hormone, and mammalian Ecd homologs have been implicated in cell cycle regulation and cancer. Why the Drosophila ecd1 mutants lack ecdysone has not been resolved. Here, we show that in Drosophila cells, Ecd directly interacts with core components of the U5 snRNP spliceosomal complex, including the conserved Prp8 protein. In accord with a function in pre-mRNA splicing, Ecd and Prp8 are cell-autonomously required for survival of proliferating cells within the larval imaginal discs. In the steroidogenic prothoracic gland, loss of Ecd or Prp8 prevents splicing of a large intron from CYP307A2/spookier (spok pre-mRNA, thus eliminating this essential ecdysone-biosynthetic enzyme and blocking the entry to metamorphosis. Human Ecd (hEcd can substitute for its missing fly ortholog. When expressed in the Ecd-deficient prothoracic gland, hEcd re-establishes spok pre-mRNA splicing and protein expression, restoring ecdysone synthesis and normal development. Our work identifies Ecd as a novel pre-mRNA splicing factor whose function has been conserved in its human counterpart. Whether the role of mammalian Ecd in cancer involves pre-mRNA splicing remains to be discovered.

  16. Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy.

    Science.gov (United States)

    Shiga, N; Takeshima, Y; Sakamoto, H; Inoue, K; Yokota, Y; Yokoyama, M; Matsuo, M

    1997-11-01

    The mechanism of exon skipping induced by nonsense mutations has not been well elucidated. We now report results of in vitro splicing studies which disclosed that a particular example of exon skipping is due to disruption of a splicing enhancer sequence located within the exon. A nonsense mutation (E1211X) due to a G to T transversion at the 28th nucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Becker muscular dystrophy case. Partial skipping of the exon resulted in the production of truncated dystrophin mRNA, although the consensus sequences for splicing at both ends of exon 27 were unaltered. To determine how E1211X induced exon 27 skipping, the splicing enhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. The mutant sequence containing G3839T abolished splicing enhancer activity of the wild-type purine-rich sequence for the upstream intron in this chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicking the purine-rich sequence of exon 27 also showed enhancer activity that was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a nonsense codon was created by the inserted T residue. This is the first evidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence. PMID:9410897

  17. Linking splicing to Pol II transcription stabilizes pre-mRNAs and influences splicing patterns.

    OpenAIRE

    Hicks, Martin J; Chin-Rang Yang; Matthew V Kotlajich; Hertel, Klemens J.

    2006-01-01

    RNA processing is carried out in close proximity to the site of transcription, suggesting a regulatory link between transcription and pre-mRNA splicing. Using an in vitro transcription/splicing assay, we demonstrate that an association of RNA polymerase II ( Pol II) transcription and pre-mRNA splicing is required for efficient gene expression. Pol II-synthesized RNAs containing functional splice sites are protected from nuclear degradation, presumably because the local concentration of the sp...

  18. Control of Alternative Splicing by Signal-dependent Degradation of Splicing-regulatory Proteins*S⃞

    OpenAIRE

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

    2009-01-01

    Alternative pre-mRNA splicing is a major gene expression regulatory mechanism in metazoan organisms. Proteins that bind pre-mRNA elements and control assembly of splicing complexes regulate utilization of pre-mRNA alternative splice sites. To understand how signaling pathways impact this mechanism, an RNA interference screen in Drosophila S2 cells was used to identify proteins that regulate TAF1 (TBP-associated factor 1) alternative splicing in response to activation o...

  19. Competition between Pre-mRNAs for the splicing machinery drives global regulation of splicing

    OpenAIRE

    Munding, EM; Shiue, L; Katzman, S.; Donohue, J; Ares, M

    2013-01-01

    During meiosis in yeast, global splicing efficiency increases and then decreases. Here we provide evidence that splicing improves due to reduced competition for the splicing machinery. The timing of this regulation corresponds to repression and reactivation of ribosomal protein genes (RPGs) during meiosis. In vegetative cells, RPG repression by rapamycin treatment also increases splicing efficiency. Downregulation of the RPG-dedicated transcription factor gene IFH1 genetically suppresses two ...

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

    OpenAIRE

    Liana F Lareau; Brenner, Steven E.

    2015-01-01

    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 N...

  1. Evolutionary conservation of alternative splicing in chicken

    OpenAIRE

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

    2007-01-01

    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 alternati...

  2. SPA: a probabilistic algorithm for spliced alignment.

    OpenAIRE

    Erik van Nimwegen; Nicodeme Paul; Robert Sheridan; Mihaela Zavolan

    2006-01-01

    Recent large-scale cDNA sequencing efforts show that elaborate patterns of splice variation are responsible for much of the proteome diversity in higher eukaryotes. To obtain an accurate account of the repertoire of splice variants, and to gain insight into the mechanisms of alternative splicing, it is essential that cDNAs are very accurately mapped to their respective genomes. Currently available algorithms for cDNA-to-genome alignment do not reach the necessary level of accuracy because the...

  3. Intron retention and rhythmic diel pattern regulation of carotenoid cleavage dioxygenase 2 during crocetin biosynthesis in saffron.

    Science.gov (United States)

    Ahrazem, Oussama; Rubio-Moraga, Angela; Argandoña-Picazo, Javier; Castillo, Raquel; Gómez-Gómez, Lourdes

    2016-06-01

    The carotenoid cleavage dioxygenase 2, a new member of the CCD family, catalyzes the conversion of zeaxanthin into crocetin-dialdehyde in Crocus. CCD2 is expressed in flowers, being responsible for the yellow, orange and red colorations displayed by tepals and stigma. Three CsCCD2 genes were identified in Crocus sativus, the longest contains ten exons and the shorter is a truncated copy with no introns and which lacks one exon sequence. Analysis of RNA-seq datasets of three developmental stages of saffron stigma allowed the determination of alternative splicing in CsCCD2, being intron retention (IR) the prevalent form of alternative splicing in CsCCD2. Further, high IR was observed in tissues that do not accumulate crocetin. The analysis of one CsCCD2 promoter showed cis-regulatory motifs involved in the response to light, temperature, and circadian regulation. The light and circadian regulation are common elements shared with the previously characterized CsLycB2a promoter, and these shared common cis-acting elements may represent binding sites for transcription factors responsible for co-regulation of these genes during the development of the stigma in saffron. A daily coordinated rhythmic regulation for CsCCD2 and CsLycB2a was observed, with higher levels of mRNA occurring at low temperatures during darkness, confirming the results obtained in the in silico promoter analysis. In addition, to the light and temperature dependent regulation of CsCCD2 expression, the apocarotenoid β-cyclocitral up-regulated CsCCD2 expression and could acts as a mediator of chromoplast-to-nucleus signalling, coordinating the expression of CsCCD2 with the developmental state of the chromoplast in the developing stigma. PMID:27071403

  4. Co-option of the piRNA Pathway for Germline-Specific Alternative Splicing of C. elegans TOR

    Directory of Open Access Journals (Sweden)

    Sergio Barberán-Soler

    2014-09-01

    Full Text Available Many eukaryotic genes contain embedded antisense transcripts and repetitive sequences of unknown function. We report that male germline-specific expression of an antisense transcript contained in an intron of C. elegans Target of Rapamycin (TOR, let-363 is associated with (1 accumulation of endo-small interfering RNAs (siRNAs against an embedded Helitron transposon and (2 activation of an alternative 3′ splice site of TOR. The germline-specific Argonaute proteins PRG-1 and CSR-1, which participate in self/nonself RNA recognition, antagonistically regulate the generation of these endo-siRNAs, TOR mRNA levels, and 3′ splice-site selection. Supply of exogenous double-stranded RNA against the region of sense/antisense overlap reverses changes in TOR expression and splicing and suppresses the progressive multigenerational sterility phenotype of prg-1 mutants. We propose that recognition of a “nonself” intronic transposon by endo-siRNAs/the piRNA system provides physiological regulation of expression and alternative splicing of a host gene that, in turn, contributes to the maintenance of germline function across generations.

  5. IntDb: A comprehensive database for classified introns of saccharomyces & human

    OpenAIRE

    Mohanty, Subhalaxmi; Nizam, Amouda; Biswal, Monalisha

    2012-01-01

    Introns (intra-genic) are non-coding regions of several eukaryotic genes. However, their role in regulation of transcription, embryonic development, stimulate gene (HEG) is apparent in recent years. Thus current research focuses on mutation in introns and their influence in causing various diseases. Though many available intron databases like YIDB, IDB, ExInt, GISSD, FUGOID, etc. discusses on various aspects of introns but none of them have classified the introns where identification of start...

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

    International Nuclear Information System (INIS)

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

  7. A large insertion in intron 2 of the TYRP1 gene associated with American Palomino phenotype in American mink.

    Science.gov (United States)

    Cirera, Susanna; Markakis, Marios Nektarios; Kristiansen, Thea; Vissenberg, Kris; Fredholm, Merete; Christensen, Knud; Anistoroaei, Razvan

    2016-04-01

    A number of American mink phenotypes display a range of brownish colours. One of these phenotypes, namely American Palomino (b (P) b (P) ) (AP) has been found to be associated with the tyrosinase-related protein 1 (TYRP1) gene by genotyping microsatellite markers in one sire family. Trials for amplifying the genomic DNA and cDNA at the beginning of intron 2 of AP TYRP1 revealed the presence of a large insertion of approximately eight kb. The insertion most likely disrupts different elements necessary for the splicing of intron 2 of the TYRP1 gene. In AP RNAseq data indicate, however, the presence of the wild-type (wt) transcript at very low levels and Western blot reveals three products when using an antibody raised against middle part of the TYRP1 protein. One individual from another brown mink phenotype-commercially named Dawn-was also investigated at the molecular level by long-range PCR and the same size insertion appears to be present. By this we suggest that certain modifiers of TYRP1 would induce different brown colour degradation, which results in at least two different phases of brown. PMID:26886941

  8. Alternative mRNA Splicing: Control by Combination

    OpenAIRE

    Mabon, Stephen A; Tom Misteli

    2005-01-01

    Alternative splicing in mammalian cells has been suggested to be largely controlled by combinatorial binding of basal splicing factors to pre-mRNA templates. This model predicts that distinct sets of pre-mRNA splicing factors are associated with alternatively spliced transcripts. However, no experimental evidence for differential recruitment of splicing factors to transcripts with distinct splicing fates is available. Here we have used quantitative single-cell imaging to test this key predict...

  9. Splicing mutation of a gene within the Duchenne muscular dystrophy family.

    Science.gov (United States)

    Zhu, Y B; Gan, J H; Luo, J W; Zheng, X Y; Wei, S C; Hu, D

    2016-01-01

    The aim of this study was to identify the mutation site and phenotype of the Duchenne muscular dystrophy (DMD) gene in a DMD family. The DMD gene is by far the largest known gene in humans. Up to 34% of the point mutations reported to date affect splice sites of the DMD gene. However, no hotspot mutation has been reported. Capture sequencing of second-generation exons was used to investigate the DMD gene in a proband. Sanger sequencing was performed for mutation scanning in eight family members. Scale-invariant feature transform and PolyPhen were applied to predict the functional impact of protein mutations. A hemizygous splicing mutation IVS44ds +1G-A (c.6438 +1G>A) that induces abnormal splicing variants during late transcription and produces abnormal proteins was located in intron 44. Four missense mutations (p.Arg2937Gln, p.Asp882Gly, p.Lys2366Gln, and p.Arg1745His) that are known multiple-polymorphic sites were found in the coding region of the DMD gene. A heterozygous c.6438+1G>A mutation was detected on the X chromosome of the proband's mother and maternal grandmother. PMID:27421007

  10. Changes in Alternative Splicing in Apis Mellifera Bees Fed Apis Cerana Royal Jelly

    Directory of Open Access Journals (Sweden)

    Shi Yuan Yuan

    2014-12-01

    Full Text Available The Western honey bee (Apis mellifera is a social insect characterized by caste differentiation in which the queen bee and worker bees display marked differences in morphology, behavior, reproduction, and longevity despite their identical genomes. The main causative factor in caste differentiation is the food fed to queen larvae, termed royal jelly (RJ. Alternative splicing (AS is an important RNA-mediated post-transcriptional process in eukaryotes. Here we report AS changes in A. mellifera after being fed either A. mellifera RJ or A. cerana RJ. The results demonstrated that the RJ type affected 4 types of AS in adult A. mellifera: exon skipping, intron retention, alternative 5’ splice sites, and alternative 3’splice sites. After feeding with A. cerana RJ, AS occurred in many genes in adult A. mellifera that encode proteins involved in development, growth, the tricarboxylic acid cycle, and substance metabolism. This study provides the first evidence that heterospecific RJ can influence the AS of many genes related to honey bee development and growth.

  11. A rare sequence variant in intron 1 of THAP1 is associated with primary dystonia.

    Science.gov (United States)

    Vemula, Satya R; Xiao, Jianfeng; Zhao, Yu; Bastian, Robert W; Perlmutter, Joel S; Racette, Brad A; Paniello, Randal C; Wszolek, Zbigniew K; Uitti, Ryan J; Van Gerpen, Jay A; Hedera, Peter; Truong, Daniel D; Blitzer, Andrew; Rudzińska, Monika; Momčilović, Dragana; Jinnah, Hyder A; Frei, Karen; Pfeiffer, Ronald F; LeDoux, Mark S

    2014-05-01

    Although coding variants in THAP1 have been causally associated with primary dystonia, the contribution of noncoding variants remains uncertain. Herein, we examine a previously identified Intron 1 variant (c.71+9C>A, rs200209986). Among 1672 subjects with mainly adult-onset primary dystonia, 12 harbored the variant in contrast to 1/1574 controls (P A variant was associated with an elevated ratio of Isoform 1 (NM_018105) to Isoform 2 (NM_199003) in leukocytes. In silico and minigene analyses indicated that c.71+9C>A alters THAP1 splicing. Lymphoblastoid cells harboring the c.71+9C>A variant showed extensive apoptosis with relatively fewer cells in the G2 phase of the cell cycle. Differentially expressed genes from lymphoblastoid cells revealed that the c.71+9C>A variant exerts effects on DNA synthesis, cell growth and proliferation, cell survival, and cytotoxicity. In aggregate, these data indicate that THAP1 c.71+9C>A is a risk factor for adult-onset primary dystonia. PMID:24936516

  12. A rare sequence variant in intron 1 of THAP1 is associated with primary dystonia

    Science.gov (United States)

    Vemula, Satya R; Xiao, Jianfeng; Zhao, Yu; Bastian, Robert W; Perlmutter, Joel S; Racette, Brad A; Paniello, Randal C; Wszolek, Zbigniew K; Uitti, Ryan J; Van Gerpen, Jay A; Hedera, Peter; Truong, Daniel D; Blitzer, Andrew; Rudzińska, Monika; Momčilović, Dragana; Jinnah, Hyder A; Frei, Karen; Pfeiffer, Ronald F; LeDoux, Mark S

    2014-01-01

    Although coding variants in THAP1 have been causally associated with primary dystonia, the contribution of noncoding variants remains uncertain. Herein, we examine a previously identified Intron 1 variant (c.71+9C>A, rs200209986). Among 1672 subjects with mainly adult-onset primary dystonia, 12 harbored the variant in contrast to 1/1574 controls (P A variant was associated with an elevated ratio of Isoform 1 (NM_018105) to Isoform 2 (NM_199003) in leukocytes. In silico and minigene analyses indicated that c.71+9C>A alters THAP1 splicing. Lymphoblastoid cells harboring the c.71+9C>A variant showed extensive apoptosis with relatively fewer cells in the G2 phase of the cell cycle. Differentially expressed genes from lymphoblastoid cells revealed that the c.71+9C>A variant exerts effects on DNA synthesis, cell growth and proliferation, cell survival, and cytotoxicity. In aggregate, these data indicate that THAP1 c.71+9C>A is a risk factor for adult-onset primary dystonia. PMID:24936516

  13. Identification and characterization of NAGNAG alternative splicing in the moss Physcomitrella patens

    Directory of Open Access Journals (Sweden)

    Bolte Kathrin

    2010-04-01

    Full Text Available Abstract Background Alternative splicing (AS involving tandem acceptors that are separated by three nucleotides (NAGNAG is an evolutionarily widespread class of AS, which is well studied in Homo sapiens (human and Mus musculus (mouse. It has also been shown to be common in the model seed plants Arabidopsis thaliana and Oryza sativa (rice. In one of the first studies involving sequence-based prediction of AS in plants, we performed a genome-wide identification and characterization of NAGNAG AS in the model plant Physcomitrella patens, a moss. Results Using Sanger data, we found 295 alternatively used NAGNAG acceptors in P. patens. Using 31 features and training and test datasets of constitutive and alternative NAGNAGs, we trained a classifier to predict the splicing outcome at NAGNAG tandem splice sites (alternative splicing, constitutive at the first acceptor, or constitutive at the second acceptor. Our classifier achieved a balanced specificity and sensitivity of ≥ 89%. Subsequently, a classifier trained exclusively on data well supported by transcript evidence was used to make genome-wide predictions of NAGNAG splicing outcomes. By generation of more transcript evidence from a next-generation sequencing platform (Roche 454, we found additional evidence for NAGNAG AS, with altogether 664 alternative NAGNAGs being detected in P. patens using all currently available transcript evidence. The 454 data also enabled us to validate the predictions of the classifier, with 64% (80/125 of the well-supported cases of AS being predicted correctly. Conclusion NAGNAG AS is just as common in the moss P. patens as it is in the seed plants A. thaliana and O. sativa (but not conserved on the level of orthologous introns, and can be predicted with high accuracy. The most informative features are the nucleotides in the NAGNAG and in its immediate vicinity, along with the splice sites scores, as found earlier for NAGNAG AS in animals. Our results suggest that the

  14. Structural Studies of RNA Helicases Involved in Eukaryotic Pre-mRNA Splicing, Ribosome Biogenesis, and Translation Initiation

    DEFF Research Database (Denmark)

    He, Yangzi

    Ribonucleic acids (RNAs) take centre stage in gene expression. In eukaryotes, most RNAs are transcribed as precursors, and these precursors are co- or post-transcriptionally processed and assemble with particular proteins to form ribonucleoproteins (RNPs). Mature RNPs participate in various gene...... expression events, are then subject to recycling, disassembly or degradation. RNA helicases are highly conserved enzymes that use ATP to bind or remodel RNA or RNPs. They function in nearly every aspect of eukaryotic RNA metabolism. The spliceosome catalyzes pre-mRNAs splicing, which removes introns and...

  15. Correlations between recombination rate and intron distributions along chromosomes of C.elegans

    Institute of Scientific and Technical Information of China (English)

    Hong Li; Guoqing Liu; Xuhua Xia

    2009-01-01

    Generally speaking,the intron size positively correlates with recombination rate in Caenorhabditis elegans genome.Here,we analyze the correlations between recombination rate and some measures of different intron lengths so as to know whether the recombination influences the introns of different lengths in the same way.Results show that the correlation between the recombination rate and the percentage of short introns(<100 bp)is negative,but the correlation between the recombination rate and the percentage of introns that are larger than 500 bp is positive.Average intron length correlates positively with the recombination rate for introns whose length is in the range of 100-1000 bp.We speculate that the recombination mainly exerts impact on introns whose length ranges from 100-1000 bp.We also show that the average intron number per gene correlates negatively with the recombination rate.

  16. Analysis of intron sequence features associated with transcriptional regulation in human genes.

    Directory of Open Access Journals (Sweden)

    Huimin Li

    Full Text Available Although some preliminary work has revealed the potential transcriptional regulatory function of the introns in eukaryotes, additional evidences are needed to support this conjecture. In this study, we perform systemic analyses of the sequence characteristics of human introns. The results show that the first introns are generally longer and C, G and their dinucleotide compositions are over-represented relative to other introns, which are consistent with the previous findings. In addition, some new phenomena concerned with transcriptional regulation are found: i the first introns are enriched in CpG islands; and ii the percentages of the first introns containing TATA, CAAT and GC boxes are relatively higher than other position introns. The similar features of introns are observed in tissue-specific genes. The results further support that the first introns of human genes are likely to be involved in transcriptional regulation, and give an insight into the transcriptional regulatory regions of genes.

  17. Post-transcriptional Repair of a Split Heat Shock Protein 90 Gene by mRNA trans-Splicing*♦

    Science.gov (United States)

    Nageshan, Rishi Kumar; Roy, Nainita; Hehl, Adrian B.; Tatu, Utpal

    2011-01-01

    Heat shock protein 90 participates in diverse biological processes ranging from protein folding, cell cycle, signal transduction and development to evolution in all eukaryotes. It is also critically involved in regulating growth of protozoa such as Dictyostelium discoideum, Leishmania donovani, Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma evansi. Selective inhibition of Hsp90 has also been explored as an intervention strategy against important human diseases such as cancer, malaria, or trypanosomiasis. Giardia lamblia, a simple protozoan parasite of humans and animals, is an important cause of diarrheal disease with significant morbidity and some mortality in tropical countries. Here we show that the G. lamblia cytosolic hsp90 (glhsp90) is split in two similar sized fragments located 777 kb apart on the same scaffold. Intrigued by this unique arrangement, which appears to be specific for the Giardiinae, we have investigated the biosynthesis of GlHsp90. We used genome sequencing to confirm the split nature of the giardial hsp90. However, a specific antibody raised against the peptide detected a product with a mass of about 80 kDa, suggesting a post-transcriptional rescue of the genomic defect. We show evidence for the joining of the two independent Hsp90 transcripts in-trans to one long mature mRNA presumably by RNA splicing. The splicing junction carries hallmarks of classical cis-spliced introns, suggesting that the regular cis-splicing machinery may be sufficient for repair of the open reading frame. A complementary 26-nt sequence in the “intron” regions adjacent to the splice sites may assist in positioning the two pre-mRNAs for processing. This is the first example of post-transcriptional rescue of a split gene by trans-splicing. PMID:21209094

  18. Genome-wide analysis of alternative splicing of pre-mRNA under salt stress in Arabidopsis

    KAUST Repository

    Ding, Feng

    2014-06-04

    Background: Alternative splicing (AS) of precursor mRNA (pre-mRNA) is an important gene regulation process that potentially regulates many physiological processes in plants, including the response to abiotic stresses such as salt stress.Results: To analyze global changes in AS under salt stress, we obtained high-coverage (~200 times) RNA sequencing data from Arabidopsis thaliana seedlings that were treated with different concentrations of NaCl. We detected that ~49% of all intron-containing genes were alternatively spliced under salt stress, 10% of which experienced significant differential alternative splicing (DAS). Furthermore, AS increased significantly under salt stress compared with under unstressed conditions. We demonstrated that most DAS genes were not differentially regulated by salt stress, suggesting that AS may represent an independent layer of gene regulation in response to stress. Our analysis of functional categories suggested that DAS genes were associated with specific functional pathways, such as the pathways for the responses to stresses and RNA splicing. We revealed that serine/arginine-rich (SR) splicing factors were frequently and specifically regulated in AS under salt stresses, suggesting a complex loop in AS regulation for stress adaptation. We also showed that alternative splicing site selection (SS) occurred most frequently at 4 nucleotides upstream or downstream of the dominant sites and that exon skipping tended to link with alternative SS.Conclusions: Our study provided a comprehensive view of AS under salt stress and revealed novel insights into the potential roles of AS in plant response to salt stress. 2014 Ding et al.; licensee BioMed Central Ltd.

  19. Pax258 and Pax6 alternative splicing events in basal chordates and vertebrates: a focus on paired box domain

    Directory of Open Access Journals (Sweden)

    Peter eFabian

    2015-07-01

    Full Text Available Paired box transcription factors play important role in development and tissue morphogenesis. The number of Pax homologs varies among species studied so far, due to genome and gene duplications that have affected PAX family to a great extent. Based on sequence similarity and functional domains, four Pax classes have been identified in chordates, namely Pax1/9, Pax2/5/8, Pax3/7 and Pax4/6. Numerous splicing events have been reported mainly for Pax2/5/8 and Pax6 genes. Of significant interest are those events that lead to Pax proteins with presumed novel properties, such as altered DNA-binding or transcriptional activity. In the current study, a thorough analysis of Pax2/5/8 splicing events from cephalochordate and vertebrates was performed. We focused more on Pax2/5/8 and Pax6 splicing events in which the paired domain is involved. Three new splicing events were identified in Oryzias latipes, one of which seems to be conserved in Acanthomorphata. Using representatives from deuterostome and protostome phyla, a comparative analysis of the Pax6 exon-intron structure of the paired domain was performed, during an attempt to estimate the time of appearance of the Pax6(5a mRNA isoform. As shown in our analysis, this splicing event is absent in basal chordates and is characteristic of Gnathostomata. Moreover, expression pattern of alternative spliced variants was compared between basal chordates and fish species. In summary, our data indicate expansion of alternative mRNA variants in paired box region of Pax2/5/8 and Pax6 genes during the course of vertebrate evolution.

  20. Intron V, not intron I of human thrombopoietin, improves expression in the milk of transgenic mice regulated by goat beta-casein promoter

    OpenAIRE

    Yan Li; Hu Hao; Mingqian Zhou; Hongwei Zhou; Jianbin Ye; Lijun Ning; Yunshan Ning

    2015-01-01

    Introns near 5′ end of genes generally enhance gene expression because of an enhancer /a promoter within their sequence or as intron-mediated enhancement. Surprisingly, our previous experiments found that the vector containing the last intron (intron V) of human thromobopoietin (hTPO) expressed higher hTPO in cos-1 cell than the vector containing intron I regulated by cytomegalovirus promoter. Moreover, regulated by 1.0 kb rat whey acidic protein promoter, hTPO expression was higher in transg...

  1. FgPrp4 Kinase Is Important for Spliceosome B-Complex Activation and Splicing Efficiency in Fusarium graminearum

    Science.gov (United States)

    Jiang, Cong; Li, Yang; Li, Chaohui; Liu, Huiquan; Kang, Zhensheng; Xu, Jin-Rong

    2016-01-01

    PRP4 encodes the only kinase among the spliceosome components. Although it is an essential gene in the fission yeast and other eukaryotic organisms, the Fgprp4 mutant was viable in the wheat scab fungus Fusarium graminearum. Deletion of FgPRP4 did not block intron splicing but affected intron splicing efficiency in over 60% of the F. graminearum genes. The Fgprp4 mutant had severe growth defects and produced spontaneous suppressors that were recovered in growth rate. Suppressor mutations were identified in the PRP6, PRP31, BRR2, and PRP8 orthologs in nine suppressor strains by sequencing analysis with candidate tri-snRNP component genes. The Q86K mutation in FgMSL1 was identified by whole genome sequencing in suppressor mutant S3. Whereas two of the suppressor mutations in FgBrr2 and FgPrp8 were similar to those characterized in their orthologs in yeasts, suppressor mutations in Prp6 and Prp31 orthologs or FgMSL1 have not been reported. Interestingly, four and two suppressor mutations identified in FgPrp6 and FgPrp31, respectively, all are near the conserved Prp4-phosphorylation sites, suggesting that these mutations may have similar effects with phosphorylation by Prp4 kinase. In FgPrp31, the non-sense mutation at R464 resulted in the truncation of the C-terminal 130 aa region that contains all the conserved Prp4-phosphorylation sites. Deletion analysis showed that the N-terminal 310-aa rich in SR residues plays a critical role in the localization and functions of FgPrp4. We also conducted phosphoproteomics analysis with FgPrp4 and identified S289 as the phosphorylation site that is essential for its functions. These results indicated that FgPrp4 is critical for splicing efficiency but not essential for intron splicing, and FgPrp4 may regulate pre-mRNA splicing by phosphorylation of other components of the tri-snRNP although itself may be activated by phosphorylation at S289. PMID:27058959

  2. FgPrp4 Kinase Is Important for Spliceosome B-Complex Activation and Splicing Efficiency in Fusarium graminearum.

    Science.gov (United States)

    Gao, Xuli; Jin, Qiaojun; Jiang, Cong; Li, Yang; Li, Chaohui; Liu, Huiquan; Kang, Zhensheng; Xu, Jin-Rong

    2016-04-01

    PRP4 encodes the only kinase among the spliceosome components. Although it is an essential gene in the fission yeast and other eukaryotic organisms, the Fgprp4 mutant was viable in the wheat scab fungus Fusarium graminearum. Deletion of FgPRP4 did not block intron splicing but affected intron splicing efficiency in over 60% of the F. graminearum genes. The Fgprp4 mutant had severe growth defects and produced spontaneous suppressors that were recovered in growth rate. Suppressor mutations were identified in the PRP6, PRP31, BRR2, and PRP8 orthologs in nine suppressor strains by sequencing analysis with candidate tri-snRNP component genes. The Q86K mutation in FgMSL1 was identified by whole genome sequencing in suppressor mutant S3. Whereas two of the suppressor mutations in FgBrr2 and FgPrp8 were similar to those characterized in their orthologs in yeasts, suppressor mutations in Prp6 and Prp31 orthologs or FgMSL1 have not been reported. Interestingly, four and two suppressor mutations identified in FgPrp6 and FgPrp31, respectively, all are near the conserved Prp4-phosphorylation sites, suggesting that these mutations may have similar effects with phosphorylation by Prp4 kinase. In FgPrp31, the non-sense mutation at R464 resulted in the truncation of the C-terminal 130 aa region that contains all the conserved Prp4-phosphorylation sites. Deletion analysis showed that the N-terminal 310-aa rich in SR residues plays a critical role in the localization and functions of FgPrp4. We also conducted phosphoproteomics analysis with FgPrp4 and identified S289 as the phosphorylation site that is essential for its functions. These results indicated that FgPrp4 is critical for splicing efficiency but not essential for intron splicing, and FgPrp4 may regulate pre-mRNA splicing by phosphorylation of other components of the tri-snRNP although itself may be activated by phosphorylation at S289. PMID:27058959

  3. Alternative Splicing and Differential Expression of Two Transcripts of Nicotine Adenine Dinucleotide Phosphate Oxidase B Gene from Zea mays

    Institute of Scientific and Technical Information of China (English)

    Fan Lin; Yun Zhang; Ming-Yi Jiang

    2009-01-01

    With the exception of rice, little is known about the existence of respiratory burst oxidase homolog (rboh) gene in cereals. The present study reports the cloning and analysis of a novel rboh gene, termed ZmrbohB, from maize (Zea mays L.). The full-length cDNA of ZmrbohB encodes a 942 amino acid protein containing all of the respiratory burst oxidase homolog catalytically critical motifs.Altemative splicing of ZmrbohB has generated two transcript isoforms, ZmrbohB-α and -β. Spliced transcript ZmrbohB-β retains an unspliced intron 11 that carries a premature termination codon and probably leads to nonsense-mediated mRNA decay. Expression analysis showed that two splice isoforms were differentially expressed in various tissues and at different developmental stages, and the major product was ZmrbohB-α. The transcripts of ZmrbohB-α accumulated markedly when the maize seedlings were subjected to various abiotic stimuli, such as wounding, cold (4℃), heat (40℃), UV and salinity stress. In addition, several abiotic stimuli also affected the alternative splicing pattern of ZmrbohB except wounding. These results provide new insight into roles in the expression regulation of plant rboh genes and suggest that ZmrbohB gene may play a role in response to environmental stresses.

  4. Splicing remodels messenger ribonucleoprotein architecture via eIF4A3-dependent and -independent recruitment of exon junction complex components.

    Science.gov (United States)

    Zhang, Zuo; Krainer, Adrian R

    2007-07-10

    Pre-mRNA splicing not only removes introns and joins exons to generate spliced mRNA but also results in remodeling of the spliced messenger ribonucleoprotein, influencing various downstream events. This remodeling includes the loading of an exon-exon junction complex (EJC). It is unclear how the spliceosome recruits the EJC onto the mRNA and whether EJC formation or EJC components are required for pre-mRNA splicing. Here we immunodepleted the EJC core component eIF4A3 from HeLa cell nuclear extract and found that eIF4A3 is dispensable for pre-mRNA splicing in vitro. However, eIF4A3 is required for the splicing-dependent loading of the Y14/Magoh heterodimer onto mRNA, and this activity of human eIF4A3 is also present in the Drosophila ortholog. Surprisingly, the loading of six other EJC components was not affected by eIF4A3 depletion, suggesting that their binding to mRNA involves different or redundant pathways. Finally, we found that the assembly of the EJC onto mRNA occurs at the late stages of the splicing reaction and requires the second-step splicing and mRNA-release factor HRH1/hPrp22. The EJC-dependent and -independent recruitment of RNA-binding proteins onto mRNA suggests a role for the EJC in messenger ribonucleoprotein remodeling involving interactions with other proteins already bound to the pre-mRNA, which has implications for nonsense-mediated mRNA decay and other mRNA transactions. PMID:17606899

  5. Spliced

    DEFF Research Database (Denmark)

    Addison, Courtney Page

    2016-01-01

    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......-work stretches out from science to enlist diverse publics, social formations and the natural world in the pursuit of legitimacy....

  6. A mutation at IVS1 + 5 of the von Hippel-Lindau gene resulting in intron retention in transcripts is not pathogenic in a patient with a tongue cancer?: case report

    Directory of Open Access Journals (Sweden)

    Asakawa Takeshi

    2012-03-01

    Full Text Available Abstract Background Von Hippel-Lindau disease (VHL is a dominantly inherited familial cancer syndrome predisposing the patient to a variety of malignant and benign neoplasms, most frequently hemangioblastoma, renal cell carcinoma, pheochromocytoma, and pancreatic tumors. VHL is caused by mutations of the VHL tumor suppressor gene on the short arm of chromosome 3, and clinical manifestations develop if both alleles are inactivated according to the two-hit hypothesis. VHL mutations are more frequent in the coding region and occur occasionally in the splicing region of the gene. Previously, we reported that the loss of heterozygosity (LOH of the VHL gene is common in squamous cell carcinoma tissues of the tongue. Case Presentation We describe a case of squamous cell carcinoma in the tongue caused by a point mutation in the splicing region of the VHL gene and discuss its association with VHL disease. Sequence analysis of DNA extracted from the tumor and peripheral blood of the patient with squamous cell carcinoma revealed a heterozygous germline mutation (c. 340 + 5 G > C in the splice donor sequence in intron 1 of the VHL gene. RT-PCR analysis of the exon1/intron1 junction in RNA from tumor tissue detected an unspliced transcript. Analysis of LOH using a marker with a heterozygous mutation of nucleotides (G or C revealed a deletion of the mutant C allele in the carcinoma tissues. Conclusions The fifth nucleotide G of the splice donor site of the VHL gene is important for the efficiency of splicing at that site. The development of tongue cancer in this patient was not associated with VHL disease because the mutation occurred in only a single allele of the VHL gene and that allele was deleted in tumor cells.

  7. Alternative splicing regulation during C. elegans development: splicing factors as regulated targets.

    Directory of Open Access Journals (Sweden)

    Sergio Barberan-Soler

    2008-02-01

    Full Text Available Alternative splicing generates protein diversity and allows for post-transcriptional gene regulation. Estimates suggest that 10% of the genes in Caenorhabditis elegans undergo alternative splicing. We constructed a splicing-sensitive microarray to detect alternative splicing for 352 cassette exons and tested for changes in alternative splicing of these genes during development. We found that the microarray data predicted that 62/352 (approximately 18% of the alternative splicing events studied show a strong change in the relative levels of the spliced isoforms (>4-fold during development. Confirmation of the microarray data by RT-PCR was obtained for 70% of randomly selected genes tested. Among the genes with the most developmentally regulated alternatively splicing was the hnRNP F/H splicing factor homolog, W02D3.11 - now named hrpf-1. For the cassette exon of hrpf-1, the inclusion isoform comprises 65% of hrpf-1 steady state messages in embryos but only 0.1% in the first larval stage. This dramatic change in the alternative splicing of an alternative splicing factor suggests a complex cascade of splicing regulation during development. We analyzed splicing in embryos from a strain with a mutation in the splicing factor sym-2, another hnRNP F/H homolog. We found that approximately half of the genes with large alternative splicing changes between the embryo and L1 stages are regulated by sym-2 in embryos. An analysis of the role of nonsense-mediated decay in regulating steady-state alternative mRNA isoforms was performed. We found that 8% of the 352 events studied have alternative isoforms whose relative steady-state levels in embryos change more than 4-fold in a nonsense-mediated decay mutant, including hrpf-1. Strikingly, 53% of these alternative splicing events that are affected by NMD in our experiment are not obvious substrates for NMD based on the presence of premature termination codons. This suggests that the targeting of splicing factors

  8. Interplay between U2 snRNP and 3 prime splice factor(s) for branch point selection on human. beta. -globin pre-mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Alibert, C.; Tazi, J.; Temsamani, J.; Jeanteur, P.; Brunel, C.; Cathala, G. (URA CNRS, Montpellier (France) Universite des Sciences et Techniques du Languedoc, Montpellier (France))

    1990-01-25

    The authors investigated the interaction of U2 snRNP with the branch-3{prime} splice rite region of three human {beta}-globin pre-mRNAs carrying nearly complete (BamHI RNA), 24 nt (AvaII RNA) and 14 nt (AccI RNA) of exon 2. All supported splicing, but mRNAs yields were respectively 2 and 10 times lower for AvaII and AccI RNAs than for BamHI. Analysis of RNase T{sub 1}-resistant fragments immunoprecipitated by an anti-(U2)RNP antibody at early times of the splicing reaction showed that the protection encompasses both the branch point region and the end of the intron in BamHI and AvaII, but essentially only the branch point in AccI RNAs. Similar experiments performed at late times with an anti-Sm antibody recognizing all snRNPs showed that the end of the intron is protected in all but BamHI RNAs. These results support the conclusion that U2 snRNP binds to a fully efficient precursor (BamHI RNA) through another factor(s) recognizing the 3{prime} splice site. Either the absence of an initial contact between U2 snRNP and the factor(s) recognizing the end of the intron (AccI RNA) or the unability of this ternary complex to undergo a conformational change (AvaII RNA) could render these severely truncated precursors poor substrates. These different situations have consequences on the branch point selection itself. BamHI and AvaII RNAs use three functional branch points at early times. Later one, all three branch points are used at the same rate in AvaII, while the usual one prevails in BamHI RNAs.

  9. Frequent gain and loss of introns in fungal cytochrome b genes.

    Directory of Open Access Journals (Sweden)

    Liang-Fen Yin

    Full Text Available In this study, all available cytochrome b (Cyt b genes from the GOBASE database were compiled and the evolutionary dynamics of the Cyt b gene introns was assessed. Cyt b gene introns were frequently present in the fungal kingdom and some lower plants, but generally absent or rare in Chromista, Protozoa, and Animalia. Fungal Cyt b introns were found at 35 positions in Cyt b genes and the number of introns varied at individual positions from a single representative to 32 different introns at position 131, showing a wide and patchy distribution. Many homologous introns were present at the same position in distantly related species but absent in closely related species, suggesting that introns of the Cyt b genes were frequently lost. On the other hand, highly similar intron sequences were observed in some distantly related species rather than in closely related species, suggesting that these introns were gained independently, likely through lateral transfers. The intron loss-and-gain events could be mediated by transpositions that might have occurred between nuclear and mitochondria. Southern hybridization analysis confirmed that some introns contained repetitive sequences and might be transposable elements. An intron gain in Botryotinia fuckeliana prevented the development of QoI fungicide resistance, suggesting that intron loss-and-gain events were not necessarily beneficial to their host organisms.

  10. Naturally occuring nucleosome positioning signals in human exons and introns

    DEFF Research Database (Denmark)

    Baldi, Pierre; Brunak, Søren; Chauvin, Yves;

    1996-01-01

    We describe the structural implications of a periodic pattern found in human exons and introns by hidden Markov models. We show that exons (besides the reading frame) have a specific sequential structure in the form of a pattern with triplet consensus non-T(A/T)G, and a minimal periodicity...

  11. Aberrant splicing and drug resistance in AML.

    Science.gov (United States)

    de Necochea-Campion, Rosalia; Shouse, Geoffrey P; Zhou, Qi; Mirshahidi, Saied; Chen, Chien-Shing

    2016-01-01

    The advent of next-generation sequencing technologies has unveiled a new window into the heterogeneity of acute myeloid leukemia (AML). In particular, recurrent mutations in spliceosome machinery and genome-wide aberrant splicing events have been recognized as a prominent component of this disease. This review will focus on how these factors influence drug resistance through altered splicing of tumor suppressor and oncogenes and dysregulation of the apoptotic signaling network. A better understanding of these factors in disease progression is necessary to design appropriate therapeutic strategies recognizing specific alternatively spliced or mutated oncogenic targets. PMID:27613060

  12. ASDB: database of alternatively spliced genes

    OpenAIRE

    Dralyuk, I; Brudno, M.; Gelfand, M S; Zorn, M.; Dubchak, I.

    2000-01-01

    Version 2.1 of ASDB (Alternative Splicing Data Base) contains 1922 protein and 2486 DNA sequences. The protein entries from SWISS-PROT are joined into clusters corresponding to alternatively spliced variants of one gene. The DNA division consists of complete genes with alternative splicing mentioned or annotated in GenBank. The search engine allows one to search over SWISS-PROT and GenBank fields and then follow the links to all variants. The database can be assessed at the URL http://cbcg.ne...

  13. 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.

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

    OpenAIRE

    Topa, Hande; Honkela, Antti

    2016-01-01

    Motivation: Alternative splicing is an important mechanism in which the regions of pre-mRNAs are differentially joined in order to form different transcript isoforms. Alternative splicing is involved in the regulation of normal physiological functions but also linked to the development of diseases such as cancer. We analyse differential expression and splicing using RNA-sequencing time series in three different settings: overall gene expression levels, absolute transcript expression levels an...

  15. Intron V, not intron I of human thrombopoietin, improves expression in the milk of transgenic mice regulated by goat beta-casein promoter.

    Science.gov (United States)

    Li, Yan; Hao, Hu; Zhou, Mingqian; Zhou, Hongwei; Ye, Jianbin; Ning, Lijun; Ning, Yunshan

    2015-01-01

    Introns near 5' end of genes generally enhance gene expression because of an enhancer /a promoter within their sequence or as intron-mediated enhancement. Surprisingly, our previous experiments found that the vector containing the last intron (intron V) of human thromobopoietin (hTPO) expressed higher hTPO in cos-1 cell than the vector containing intron I regulated by cytomegalovirus promoter. Moreover, regulated by 1.0 kb rat whey acidic protein promoter, hTPO expression was higher in transgenic mice generated by intron V-TPOcDNA than in transgenic mice generated by TPOcDNA and TPOgDNA. However, it is unknown whether the enhancement of hTPO expression by intron I is decreased by uAUG7 at 5'-UTR of hTPO in vivo. Currently, we constructed vectors regulated by stronger 6.5 kb β-casein promoter, including pTPOGA (containing TPOcDNA), pTPOGB (containing TUR-TPOcDNA, TUR including exon1, intron I and non-coding exon2 of hTPO gene), pTPOGC (containing ΔTUR-TPOcDNA, nucleotides of TUR from uAUG7 to physiological AUG were deleted), pTPOGD (containing intron V-TPOcDNA) and pTPOGE (containing TPOgDNA), to evaluate the effect of intron I on hTPO expression and to further verify whether intron V enhances hTPO expression in the milk of transgenic mice. The results demonstrated that intron V, not intron I improved hTPO expression. PMID:26527459

  16. 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. PMID:25555158

  17. Expression of Cyclooxygenase-2 mRNA and Identification of Its Splice Variant in Human Myometrium Obtained from Women in Labor

    Institute of Scientific and Technical Information of China (English)

    HUANG Yinping; WAN Jingyuan; YE Duyun; WU Ping; HUANG Yanjun; ZHANG Li; ZHOU Xiaoyan; HUANG Yunfeng; YUAN Ping; ZHANG Daijuan

    2005-01-01

    In order to investigate the expression of cyclooxygenase-2 (COX-2) in human lower segments of myometrium obtained from women in labor and those not in labor and identify the splicing variant of COX-2, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of COX-2. The primers were designed and synthesized according to the sequence of rat COX-2 splice variant which was discovered firstly by us. Then the splicing variant of COX-2 in human myometrium from woman in labor was identified, cloned into vector and sequenced. The results showed that the expression of COX-2 mRNA was lower in human myometrium obtained from women who were not in labor than that in labor women and a new band of COX-2 was obtained in myometrium from labor woman. The fragment included an unspliced intron, which pitched between exons 7 and 8. It was suggested that COX-2 gene was not only expressed highly in human myometrium from woman in labor, but also produced splicing variant by alternative splicing.

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

    Directory of Open Access Journals (Sweden)

    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

  19. Hollywood: a comparative relational database of alternative splicing

    OpenAIRE

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

    2005-01-01

    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 ...

  20. HIV-1 Vpr: A Novel Role in Regulating RNA Splicing

    OpenAIRE

    Zhang, Xianfeng; Aida, Yoko

    2009-01-01

    Pre-mRNA splicing is a critical step in gene expression for metazoans. Several viral proteins regulate the splicing of pre-mRNAs through complex interactions between the virus and the host cell RNA splicing machinery. Here, we focus on a novel function of HIV-1 Vpr, that selectively inhibit cellular and viral pre-mRNA splicing, via interactions with components of functional spliceosomal complexes. This review discusses our current knowledge of how RNA splicing regulation is accomplished by Vp...

  1. A Novel Type of Splicing Enhancer Regulating Adenovirus Pre-mRNA Splicing

    OpenAIRE

    Mühlemann, Oliver; Yue, Bai-Gong; Petersen-Mahrt, Svend; Akusjärvi, Göran

    2000-01-01

    Splicing of the adenovirus IIIa pre-mRNA is subjected to a temporal regulation, such that efficient IIIa 3′ splice site usage is confined to the late phase of the infectious cycle. Here we show that IIIa pre-mRNA splicing is activated more than 200-fold in nuclear extracts prepared from late adenovirus-infected cells (Ad-NE) compared to uninfected HeLa cell nuclear extracts (HeLa-NE). In contrast, splicing of the β-globin pre-mRNA is repressed in Ad-NE. We constructed hybrid pre-mRNAs between...

  2. Competition between pre-mRNAs for the splicing machinery drives global regulation of splicing.

    Science.gov (United States)

    Munding, Elizabeth M; Shiue, Lily; Katzman, Sol; Donohue, John Paul; Ares, Manuel

    2013-08-01

    During meiosis in yeast, global splicing efficiency increases and then decreases. Here we provide evidence that splicing improves due to reduced competition for the splicing machinery. The timing of this regulation corresponds to repression and reactivation of ribosomal protein genes (RPGs) during meiosis. In vegetative cells, RPG repression by rapamycin treatment also increases splicing efficiency. Downregulation of the RPG-dedicated transcription factor gene IFH1 genetically suppresses two spliceosome mutations, prp11-1 and prp4-1, and globally restores splicing efficiency in prp4-1 cells. We conclude that the splicing apparatus is limiting and that pre-messenger RNAs compete. Splicing efficiency of a pre-mRNA therefore depends not just on its own concentration and affinity for limiting splicing factor(s), but also on those of competing pre-mRNAs. Competition between RNAs for limiting processing factors appears to be a general condition in eukaryotes for a variety of posttranscriptional control mechanisms including microRNA (miRNA) repression, polyadenylation, and splicing. PMID:23891561

  3. A site-specific endonuclease encoded by a typical archaeal intron

    DEFF Research Database (Denmark)

    Dalgaard, Jacob; Garrett, Roger Antony; Belfort, Malene

    1993-01-01

    The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing endonucleases, is capable of cleaving an intronless allele of the gene. This enzyme, I-Dmo I, is unusual among the intron endon...... of endonucleases and intron core elements and are consistent with the invasive potential of endonuclease genes....

  4. Thermal activation of a group II intron ribozyme reveals multiple conformational states.

    Science.gov (United States)

    Franzen, J S; Zhang, M; Chay, T R; Peebles, C L

    1994-09-20

    Conformational changes often accompany biological catalysis. Group II introns promote a variety of reactions in vitro that show an unusually sharp temperature dependence. This suggests that the chemical steps are accompanied by the conversion of a folded-but-inactive form to a differently folded active state. We report here the kinetic analysis of 5'-splice-junction hydrolysis (SJH) by E1:12345, a transcript containing the 5'-exon plus the first five of six intron secondary structure domains. The pseudo-first-order SJH reaction shows (1) activation by added KCl to 1.5 M; (2) cooperative activation by added MgCl2, nHill = 4.1-4.3, and [MgCl2]vmax/2 approximately 0.040 M; and (3) a rather high apparent activation energy, Ea approximately 50 kcal mol-l. In contrast, the 5'-terminal phosphodiester bond of a domain 5 transcript (GGD5) was hydrolyzed with Ea approximately 30 kcal mol-1 under SJH conditions; the 5'-GG leader dinucleotide presumably lacks secondary structure constraints. The effect of adding the chaotropic salt tetraethylammonium chloride (TEA) was also investigated. TEA reduced the melting temperatures of GGD5 and E1:12345. TEA also shifted the profile of rate versus temperature for SJH by E1:12345 toward lower temperatures without affecting the maximum rate. TEA had little effect on the rate of hydrolysis of the 5'-phosphodiester bond of GGD5. The high apparent activation enthalpy and entropy for SJH along with the effect of TEA on these parameters imply that conversion of an inactive form of E1:12345 to an active conformation accompanies enhanced occupation of the transition state as the temperature is raised to that for maximum SJH. Analytical modeling indicates that either a two-state model (open and disordered, with open being active) or a three-state model (compact, open, and disordered) could account for the temperature dependence of kSJH. However, the three-state model is clearly preferable, since it does not require that the activation parameters

  5. 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}.

  6. A Splice Defect in the EDA Gene in Dogs with an X-Linked Hypohidrotic Ectodermal Dysplasia (XLHED) Phenotype.

    Science.gov (United States)

    Waluk, Dominik P; Zur, Gila; Kaufmann, Ronnie; Welle, Monika M; Jagannathan, Vidhya; Drögemüller, Cord; Müller, Eliane J; Leeb, Tosso; Galichet, Arnaud

    2016-01-01

    X-linked hypohidrotic ectodermal dysplasia (XLHED) caused by variants in the EDA gene represents the most common ectodermal dysplasia in humans. We investigated three male mixed-breed dogs with an ectodermal dysplasia phenotype characterized by marked hypotrichosis and multifocal complete alopecia, almost complete absence of sweat and sebaceous glands, and altered dentition with missing and abnormally shaped teeth. Analysis of SNP chip genotypes and whole genome sequence data from the three affected dogs revealed that the affected dogs shared the same haplotype on a large segment of the X-chromosome, including the EDA gene. Unexpectedly, the whole genome sequence data did not reveal any nonsynonymous EDA variant in the affected dogs. We therefore performed an RNA-seq experiment on skin biopsies to search for changes in the transcriptome. This analysis revealed that the EDA transcript in the affected dogs lacked 103 nucleotides encoded by exon 2. We speculate that this exon skipping is caused by a genetic variant located in one of the large introns flanking this exon, which was missed by whole genome sequencing with the illumina short read technology. The altered EDA transcript splicing most likely causes the observed ectodermal dysplasia in the affected dogs. These dogs thus offer an excellent opportunity to gain insights into the complex splicing processes required for expression of the EDA gene, and other genes with large introns. PMID:27449516

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

    OpenAIRE

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

    1991-01-01

    The human growth-hormone-variant (hGH-V) gene normally expresses two alternatively spliced forms of mRNA--hGH-V and hGH-V2--in the placenta. hGH-V2 mRNA differs from hGH-V rDNA by the retention of intron 4 and represents approximately 15% of transcripts at term. In a survey of hGH-V gene expression in 20 placentas of gestational age 8-40 wk, we detected a single placenta that contained, in addition to the two normal hGH-V mRNA species, a set of two slightly larger hGH-V mRNAs. Sequence analys...

  8. A case of mild CHARGE syndrome associated with a splice site mutation in CHD7.

    Science.gov (United States)

    Wells, Constance; Loundon, Natalie; Garabedian, Noël; Wiener-Vacher, Sylvette; Cordier-Bouvier, Marie-Dominique; Goudeffroye, Géraldine; Attié-Bitach, Tania; Marlin, Sandrine

    2016-04-01

    CHARGE syndrome (MIM#214800) (Coloboma, Heart defect, Atresia of choanae, Retarded growth and development, Genital hypoplasia, Ear abnormalities/deafness) is caused by heterozygous mutation of CHD7 transmitted in an autosomal dominant manner. In this report, we describe a patient with bilateral hearing impairment, unusually-shaped ears, no intellectual disability and a patent ductus arteriosus. Further investigation showed abnormal semicircular canals and the presence of olfactory bulbs. He does not fulfill the Blake or the Verloes criteria for CHARGE. A de novo mutation at the donor splice site of intron 33 was identified (c.7164 + 1G > A). It is of importance to diagnose mildly affected patients for appropriate genetic counselling and to better understand the mild end of the phenotypic spectrum of CHARGE syndrome. PMID:26921530

  9. Genomewide analysis of intronic microRNAs in rice and Arabidopsis

    Indian Academy of Sciences (India)

    G. D. Yang; K. Yan; B. J. Wu; Y. H. Wang; Y. X. Gao; C. C. Zheng

    2012-12-01

    MicroRNAs (miRNAs) are potent regulators of gene transcription and posttranscriptional processes. The majority of miRNAs are localized within intronic regions of protein-coding genes (host genes) and have diverse functions in regulating important cellular processes in animals. To date, few plant intronic miRNAs have been studied functionally. Here we report a comprehensive computational analysis to characterize intronic miRNAs in rice and Arabidopsis. RT-PCR analysis confirmed that the identified intronic miRNAs were derived from the real introns of host genes. Interestingly, 13 intronic miRNAs in rice and two in Arabidopsis were located within seven clusters, of which four polycistronic clusters contain miRNAs derived from different families, suggesting that these clustered intronic miRNAs might be involved in extremely complex regulation in rice. Length analysis of miRNA-carrying introns, promoter prediction and qRT-PCR analysis results indicated that intronic miRNAs are coexpressed with their host genes. Expression pattern analysis demonstrated that host genes had a very broad expression spectrum in different stages of development, suggesting the intronic miRNAs might play an important role in plant development. This comparative genomics analysis of intronic miRNAs in rice and Arabidopsis provides new insight into the functions and regulatory mechanisms of intronic miRNAs in monocots and dicots.

  10. Tau exon 10 alternative splicing and tauopathies

    OpenAIRE

    Liu Fei; Gong Cheng-Xin

    2008-01-01

    Abstract Abnormalities of microtubule-associated protein tau play a central role in neurofibrillary degeneration in several neurodegenerative disorders that collectively called tauopathies. Six isoforms of tau are expressed in adult human brain, which result from alternative splicing of pre-mRNA generated from a single tau gene. Alternative splicing of tau exon 10 results in tau isoforms containing either three or four microtubule-binding repeats (3R-tau and 4R-tau, respectively). Approximate...

  11. Box C/D RNA guides for the ribose methylation of archaeal tRNAs. The tRNATrp intron guides the formation of two ribose-methylated nucleosides in the mature tRNATrp

    Science.gov (United States)

    d’Orval, Béatrice Clouet; Bortolin, Marie-Line; Gaspin, Christine; Bachellerie, Jean-Pierre

    2001-01-01

    Following a search of the Pyrococcus genomes for homologs of eukaryotic methylation guide small nucleolar RNAs, we have experimentally identified in Pyrococcus abyssi four novel box C/D small RNAs predicted to direct 2′-O-ribose methylations onto the first position of the anticodon in tRNALeu(CAA), tRNALeu(UAA), elongator tRNAMet and tRNATrp, respectively. Remarkably, one of them corresponds to the intron of its presumptive target, pre-tRNATrp. This intron is predicted to direct in cis two distinct ribose methylations within the unspliced tRNA precursor, not only onto the first position of the anticodon in the 5′ exon but also onto position 39 (universal tRNA numbering) in the 3′ exon. The two intramolecular RNA duplexes expected to direct methylation, which both span an exon–intron junction in pre-tRNATrp, are phylogenetically conserved in euryarchaeotes. We have experimentally confirmed the predicted guide function of the box C/D intron in halophile Haloferax volcanii by mutagenesis analysis, using an in vitro splicing/RNA modification assay in which the two cognate ribose methylations of pre-tRNATrp are faithfully reproduced. Euryarchaeal pre-tRNATrp should provide a unique system to further investigate the molecular mechanisms of RNA-guided ribose methylation and gain new insights into the origin and evolution of the complex family of archaeal and eukaryotic box C/D small RNAs. PMID:11713301

  12. Splice Junction Map of Simian Parvovirus Transcripts

    OpenAIRE

    Vashisht, Kapil; Faaberg, Kay S.; Aber, Amanda L.; Brown, Kevin E.; O’Sullivan, M. Gerard

    2004-01-01

    The transcription map of simian parvovirus (SPV), an Erythrovirus similar to Parvovirus B19, was investigated. RNA was extracted from tissues of experimentally infected cynomolgus macaques and subjected to reverse transcription-PCR with SPV-specific primers. The PCR products were cloned and sequenced to identify splice junctions. A total of 14 distinct sequences were identified as putative partial transcripts. Of these, 13 were spliced; a single unspliced transcript putatively encoded NS1. Se...

  13. 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...

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

    International Nuclear Information System (INIS)

    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 → 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 ∼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

  15. Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues [v2; ref status: indexed, http://f1000r.es/2dl

    Directory of Open Access Journals (Sweden)

    Liliana Florea

    2013-11-01

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

  16. Splicing therapy for neuromuscular disease.

    Science.gov (United States)

    Douglas, Andrew G L; Wood, Matthew J A

    2013-09-01

    Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) are two of the most common inherited neuromuscular diseases in humans. Both conditions are fatal and no clinically available treatments are able to significantly alter disease course in either case. However, by manipulation of pre-mRNA splicing using antisense oligonucleotides, defective transcripts from the DMD gene and from the SMN2 gene in SMA can be modified to once again produce protein and restore function. A large number of in vitro and in vivo studies have validated the applicability of this approach and an increasing number of preliminary clinical trials have either been completed or are under way. Several different oligonucleotide chemistries can be used for this purpose and various strategies are being developed to facilitate increased delivery efficiency and prolonged therapeutic effect. As these novel therapeutic compounds start to enter the clinical arena, attention must also be drawn to the question of how best to facilitate the clinical development of such personalised genetic therapies and how best to implement their provision. PMID:23631896

  17. Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Daniel Nilsson

    Full Text Available Trans-splicing of leader sequences onto the 5'ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5'splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT. The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5' splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.

  18. RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase.

    Science.gov (United States)

    Nottingham, Ryan M; Wu, Douglas C; Qin, Yidan; Yao, Jun; Hunicke-Smith, Scott; Lambowitz, Alan M

    2016-04-01

    Next-generation RNA sequencing (RNA-seq) has revolutionized our ability to analyze transcriptomes. Current RNA-seq methods are highly reproducible, but each has biases resulting from different modes of RNA sample preparation, reverse transcription, and adapter addition, leading to variability between methods. Moreover, the transcriptome cannot be profiled comprehensively because highly structured RNAs, such as tRNAs and snoRNAs, are refractory to conventional RNA-seq methods. Recently, we developed a new method for strand-specific RNA-seq using thermostable group II intron reverse transcriptases (TGIRTs). TGIRT enzymes have higher processivity and fidelity than conventional retroviral reverse transcriptases plus a novel template-switching activity that enables RNA-seq adapter addition during cDNA synthesis without using RNA ligase. Here, we obtained TGIRT-seq data sets for well-characterized human RNA reference samples and compared them to previous data sets obtained for these RNAs by the Illumina TruSeq v2 and v3 methods. We find that TGIRT-seq recapitulates the relative abundance of human transcripts and RNA spike-ins in ribo-depleted, fragmented RNA samples comparably to non-strand-specific TruSeq v2 and better than strand-specific TruSeq v3. Moreover, TGIRT-seq is more strand specific than TruSeq v3 and eliminates sampling biases from random hexamer priming, which are inherent to TruSeq. The TGIRT-seq data sets also show more uniform 5' to 3' gene coverage and identify more splice junctions, particularly near the 5' ends of mRNAs, than do the TruSeq data sets. Finally, TGIRT-seq enables the simultaneous profiling of mRNAs and lncRNAs in the same RNA-seq experiment as structured small ncRNAs, including tRNAs, which are essentially absent with TruSeq. PMID:26826130

  19. Identification of rare intronic variants defining novel BRCA haplotypes

    Directory of Open Access Journals (Sweden)

    Anca Negura

    2012-10-01

    Full Text Available Sanger DNA sequencing is widely used nowadays to identify germ-line alterations of cancerpredisposition genes. Molecular diagnosis of hereditary risk of breast and ovarian cancer (HBOC is mainly targetingBRCA1 and BRCA2, as carriers of deleterious mutations in any of these genes are at significantly higher risk ofdeveloping cancer than general population. Usual diagnosis performs a pre-screening step, followed by systematicsequencing of exons and exon/intron boundaries. Unfortunately, not all sequence variations found by sequencing arepathogenic. About one half of identified variants are of unclear clinical significance. Common single nucleotidepolymorphisms (SNPs are also usually detected, either heterozygous or homozygous. When systematically detected,SNPs can be used to define haplotypes, which are particularly useful in population disease studies or ethnogenomics.Frequent BRCA1 SNPs are well known, as well as associated haplotypes. Here we present some rare intronic variantsdefining novel BRCA1 haplotypes.

  20. Alternative splicing of DENND1A, a PCOS candidate gene, generates variant 2.

    Science.gov (United States)

    Tee, Meng Kian; Speek, Mart; Legeza, Balázs; Modi, Bhavi; Teves, Maria Eugenia; McAllister, Janette M; Strauss, Jerome F; Miller, Walter L

    2016-10-15

    Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by hyperandrogenism and metabolic disorders. The excess androgens may be of both ovarian and adrenal origin. PCOS has a strong genetic component, and genome-wide association studies have identified several candidate genes, notably DENND1A, which encodes connecdenn 1, involved in trafficking of endosomes. DENND1A encodes two principal variants, V1 (1009 amino acids) and V2 (559 amino acids). The androgen-producing ovarian theca cells of PCOS women over-express V2. Knockdown of V2 in these cells reduces androgen production, and overexpression of V2 in normal theca cells confers upon them a PCOS phenotype of increased androgen synthesis. We report that human adrenal NCI-H295A cells express V1 and V2 mRNA and that the V2 isoform is produced by exonization of sequences in intron 20, which generates a unique exon 20A, encoding the C-terminus of V2. As in human theca cells from normal women, forced expression of V2 in NCI-H295A cells resulted in increased abundance of CYP17A1 and CYP11A1 mRNAs. We also found genetic variation in the intronic region 330 bp upstream from exon 20A, which could have the potential to drive the selective expression of V2. There was no clear association with these variants with PCOS when we analyzed genomc DNA from normal women and women with PCOS. Using minigene expression vectors in NCI-H295A cells, this variable region did not consistently favor splicing of the V2 transcript. These findings suggest increased V2 expression in PCOS theca cells is not the result of genomic sequence variation in intron 20. PMID:27297658

  1. Both size and GC-content of minimal introns are selected in human populations.

    Directory of Open Access Journals (Sweden)

    Dapeng Wang

    Full Text Available BACKGROUND: We previously have studied the insertion and deletion polymorphism by sequencing no more than one hundred introns in a mixed human population and found that the minimal introns tended to maintain length at an optimal size. Here we analyzed re-sequenced 179 individual genomes (from African, European, and Asian populations from the data released by the 1000 Genome Project to study the size dynamics of minimal introns. PRINCIPAL FINDINGS: We not only confirmed that minimal introns in human populations are selected but also found two major effects in minimal intron evolution: (i Size-effect: minimal introns longer than an optimal size (87 nt tend to have a higher ratio of deletion to insertion than those that are shorter than the optimal size; (ii GC-effect: minimal introns with lower GC content tend to be more frequently deleted than those with higher GC content. The GC-effect results in a higher GC content in minimal introns than their flanking exons as opposed to larger introns (≥125 nt that always have a lower GC content than that of their flanking exons. We also observed that the two effects are distinguishable but not completely separable within and between populations. CONCLUSIONS: We validated the unique mutation dynamics of minimal introns in keeping their near-optimal size and GC content, and our observations suggest potentially important functions of human minimal introns in transcript processing and gene regulation.

  2. Characterization of a spliced exon product of herpes simplex type-1 latency-associated transcript in productively infected cells

    International Nuclear Information System (INIS)

    The latency-associated transcripts (LATs) of herpes simplex virus type-1 (HSV-1) are the only viral RNAs accumulating during latent infections in the sensory ganglia of the peripheral nervous system. The major form of LAT that accumulates in latently infected neurons is a 2 kb intron, spliced from a much less abundant 8.3 primary transcript. The spliced exon mRNA has been hard to detect. However, in this study, we have examined the spliced exon RNA in productively infected cells using ribonuclease protection (RPA), and quantitative RT-PCR (q-PCR) assays. We were able to detect the LAT exon RNA in productively infected SY5Y cells (a human neuronal cell line). The level of the LAT exon RNA was found to be approximately 5% that of the 2 kb intron RNA and thus is likely to be relatively unstable. Quantitative RT-PCR (q-PCR) assays were used to examine the LAT exon RNA and its properties. They confirmed that the LAT exon mRNA is present at a very low level in productively infected cells, compared to the levels of other viral transcripts. Furthermore, experiments showed that the LAT exon mRNA is expressed as a true late gene, and appears to be polyadenylated. In SY5Y cells, in contrast to most late viral transcripts, the LAT exon RNA was found to be mainly nuclear localized during the late stage of a productive infection. Interestingly, more LAT exon RNA was found in the cytoplasm in differentiated compared to undifferentiated SY5Y cells, suggesting the nucleocytoplasmic distribution of the LAT exon RNA and its related function may be influenced by the differentiation state of cells

  3. A global analysis of C. elegans trans-splicing

    OpenAIRE

    Allen, Mary Ann; Hillier, LaDeana W.; Waterston, Robert H.; Blumenthal, Thomas

    2011-01-01

    Trans-splicing of one of two short leader RNAs, SL1 or SL2, occurs at the 5′ ends of pre-mRNAs of many C. elegans genes. We have exploited RNA-sequencing data from the modENCODE project to analyze the transcriptome of C. elegans for patterns of trans-splicing. Transcripts of ∼70% of genes are trans-spliced, similar to earlier estimates based on analysis of far fewer genes. The mRNAs of most trans-spliced genes are spliced to either SL1 or SL2, but most genes are not trans-spliced to both, ind...

  4. 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.

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

    Science.gov (United States)

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

    1991-12-01

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

  6. Conservation of the positions of metazoan introns from sponges to humans.

    Science.gov (United States)

    Müller, Werner E G; Böhm, Markus; Grebenjuk, Vladislav A; Skorokhod, Alexander; Müller, Isabel M; Gamulin, Vera

    2002-08-01

    Sponges (phylum Porifera) are the phylogenetic oldest Metazoa still extant. They can be considered as reference animals (Urmetazoa) for the understanding of the evolutionary processes resulting in the creation of Metazoa in general and also for the metazoan gene organization in particular. In the marine sponge Suberites domuncula, genes encoding p38 and JNK kinases contain nine and twelve introns, respectively. Eight introns in both genes share the same positions and the identical phases. One p38 intron slipped for six bases and the JNK gene has three more introns. However, the sequences of the introns are not conserved and the introns in JNK gene are generally much longer. Introns interrupt most of the conserved kinase subdomains I-XI and are found in all three phases (0, 1 and 2). We analyzed in details p38 and JNK genes from human, Caenorhabditis elegans and Drosophila melanogaster and found in most genes introns at the positions identical to those in sponge genes. The exceptions are two p38 genes from D. melanogaster that have lost all introns in the coding sequence. The positions of 11 introns in each of four human p38 genes are fully conserved and ten introns occupy identical positions as the introns in sponge p38 or JNK genes. The same is true for nine, out of ten introns in the human JNK-1 gene. The introns in human p38 and JNK genes are on average more than ten times longer than corresponding introns in sponges. It was proposed that yeast HOG1-like kinases (from i.e. Saccharomyces cerevisiae and Emericella nidulans) and metazoan p38 and JNK kinases are orthologues. p38 and JNK genes were created after the split from fungi by the duplication and diversification of the HOG1-like progenitor gene. Our results further support the common origin of p38 and JNK genes and speak in favor of a very early time of duplication. The ancestral gene contained at least ten introns, which are still present at the very conserved positions in p38 and JNK genes of extant

  7. Cholesteryl Ester Transfer Protein (CETP polymorphisms affect mRNA splicing, HDL levels, and sex-dependent cardiovascular risk.

    Directory of Open Access Journals (Sweden)

    Audrey C Papp

    Full Text Available Polymorphisms in and around the Cholesteryl Ester Transfer Protein (CETP gene have been associated with HDL levels, risk for coronary artery disease (CAD, and response to therapy. The mechanism of action of these polymorphisms has yet to be defined. We used mRNA allelic expression and splice isoform measurements in human liver tissues to identify the genetic variants affecting CETP levels. Allelic CETP mRNA expression ratios in 56 human livers were strongly associated with several variants 2.5-7 kb upstream of the transcription start site (e.g., rs247616 p = 6.4 × 10(-5, allele frequency 33%. In addition, a common alternatively spliced CETP isoform lacking exon 9 (Δ9, has been shown to prevent CETP secretion in a dominant-negative manner. The Δ 9 expression ranged from 10 to 48% of total CETP mRNA in 94 livers. Increased formation of this isoform was exclusively associated with an exon 9 polymorphism rs5883-C>T (p = 6.8 × 10(-10 and intron 8 polymorphism rs9930761-T>C (5.6 × 10(-8 (in high linkage disequilibrium with allele frequencies 6-7%. rs9930761 changes a key splicing branch point nucleotide in intron 8, while rs5883 alters an exonic splicing enhancer sequence in exon 9.The effect of these polymorphisms was evaluated in two clinical studies. In the Whitehall II study of 4745 subjects, both rs247616 and rs5883T/rs9930761C were independently associated with increased HDL-C levels in males with similar effect size (rs247616 p = 9.6 × 10(-28 and rs5883 p = 8.6 × 10(-10, adjusted for rs247616. In an independent multiethnic US cohort of hypertensive subjects with CAD (INVEST-GENE, rs5883T/rs9930761C alone were significantly associated with increased incidence of MI, stroke, and all-cause mortality in males (rs5883: OR 2.36 (CI 1.29-4.30, p = 0.005, n = 866. These variants did not reach significance in females in either study. Similar to earlier results linking low CETP activity with poor outcomes in males, our results suggest genetic, sex

  8. Cholesteryl Ester Transfer Protein (CETP) polymorphisms affect mRNA splicing, HDL levels, and sex-dependent cardiovascular risk.

    Science.gov (United States)

    Papp, Audrey C; Pinsonneault, Julia K; Wang, Danxin; Newman, Leslie C; Gong, Yan; Johnson, Julie A; Pepine, Carl J; Kumari, Meena; Hingorani, Aroon D; Talmud, Philippa J; Shah, Sonia; Humphries, Steve E; Sadee, Wolfgang

    2012-01-01

    Polymorphisms in and around the Cholesteryl Ester Transfer Protein (CETP) gene have been associated with HDL levels, risk for coronary artery disease (CAD), and response to therapy. The mechanism of action of these polymorphisms has yet to be defined. We used mRNA allelic expression and splice isoform measurements in human liver tissues to identify the genetic variants affecting CETP levels. Allelic CETP mRNA expression ratios in 56 human livers were strongly associated with several variants 2.5-7 kb upstream of the transcription start site (e.g., rs247616 p = 6.4 × 10(-5), allele frequency 33%). In addition, a common alternatively spliced CETP isoform lacking exon 9 (Δ9), has been shown to prevent CETP secretion in a dominant-negative manner. The Δ 9 expression ranged from 10 to 48% of total CETP mRNA in 94 livers. Increased formation of this isoform was exclusively associated with an exon 9 polymorphism rs5883-C>T (p = 6.8 × 10(-10)) and intron 8 polymorphism rs9930761-T>C (5.6 × 10(-8)) (in high linkage disequilibrium with allele frequencies 6-7%). rs9930761 changes a key splicing branch point nucleotide in intron 8, while rs5883 alters an exonic splicing enhancer sequence in exon 9.The effect of these polymorphisms was evaluated in two clinical studies. In the Whitehall II study of 4745 subjects, both rs247616 and rs5883T/rs9930761C were independently associated with increased HDL-C levels in males with similar effect size (rs247616 p = 9.6 × 10(-28) and rs5883 p = 8.6 × 10(-10), adjusted for rs247616). In an independent multiethnic US cohort of hypertensive subjects with CAD (INVEST-GENE), rs5883T/rs9930761C alone were significantly associated with increased incidence of MI, stroke, and all-cause mortality in males (rs5883: OR 2.36 (CI 1.29-4.30), p = 0.005, n = 866). These variants did not reach significance in females in either study. Similar to earlier results linking low CETP activity with poor outcomes in males, our results suggest genetic, sex

  9. The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis.

    Science.gov (United States)

    Haïli, Nawel; Planchard, Noelya; Arnal, Nadège; Quadrado, Martine; Vrielynck, Nathalie; Dahan, Jennifer; des Francs-Small, Catherine Colas; Mireau, Hakim

    2016-01-01

    Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5' and 3' extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5' processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria. PMID:26537562

  10. Single nucleotide polymorphisms in intron 1 and intron 2 of Larimichthys crocea growth hormone gene are correlated with growth traits

    Institute of Scientific and Technical Information of China (English)

    NI Jing; YOU Feng; XU Jianhe; XU Dongdong; WEN Aiyun; WU Zhihao; XU Yongli; ZHANG Peijun

    2012-01-01

    The growth hormone gene (GH) affects animal growth and is a potential target for genetic studies of variation related to growth traits.In this study,we analyzed single nucleotide polymorphisms (SNPs) in GH intron regions and their associations with growth traits in large yellow croaker,Larimichthys crocea,from Zhejiang and Fujian stocks.The results of PCR-single strand conformation polymorphism showed two haplotypes of intron 1,named AA and AB genotypes,in Zhejiang stock.AB exhibited an SNP at position 196 (G→A) that was negatively correlated with body height and positively correlated with standard length/body height (P≤0.05).Two different genotypes,CC and CD,were identified in intron 2 in Fujian stock,with CD showing an SNP at position 692 (T→C).The CD genotype had a significantly positive correlation with both weight and total length (P≤0.01).These basic data highlight the potential for using GH as a genetic marker of fish growth in marker assisted selection.

  11. A splice mutation and mRNA decay of EXT2 provoke hereditary multiple exostoses.

    Directory of Open Access Journals (Sweden)

    Chen Tian

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

  12. Genome-wide analysis of shoot growth-associated alternative splicing in moso bamboo.

    Science.gov (United States)

    Li, Long; Hu, Tao; Li, Xueping; Mu, Shaohua; Cheng, Zhanchao; Ge, Wei; Gao, Jian

    2016-08-01

    Alternative splicing (AS) significantly enhances transcriptome complexity and is differentially regulated in a wide variety of physiological processes in plants, including shoot growth. Presently, the functional implications and conservation of AS occurrences are not well understood in the moso bamboo genome. To analyze the global changes in AS during moso bamboo shoot growth, fast-growing shoots collected at seven different heights and culms after leaf expansion were sequenced using the Illumina HiSeq™ 2000 sequencing platform. It was found that approximately 60.74 % of all genes were alternatively spliced, with intron retention (IR) being the most frequent AS event (27.43 %). Statistical analysis demonstrated that variations of AS frequency and AS types were significantly correlated with changes in gene features and gene transcriptional level. According to the phylogenetic analysis of isoform expression data and AS frequency, the bamboo shoot growth could be divided into four different growth periods, including winter bamboo shoot (S1), early growth period (S2-S5), late growth period (S6 and S7), and mature period (CK). In addition, our data also showed that the winter bamboo shoot had the highest number of AS events. Twenty-six putative Serine/arginine-rich (SR) proteins were identified, producing a total of 109 transcripts. AS events were frequently and specifically regulated by SR splicing factors throughout shoot growth, resulting in changes to the original open reading frame (ORF) and subsequently changes to conserved domains. The AS product-isoforms showed regular expression change during the whole shoot growth period, thus influencing shoot growth. All together, these data indicate that AS events are adjusted to different growth stages, providing briefness and efficient means of gene regulation. This study will provide a very useful clue for future functional analyses. PMID:27170010

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

    OpenAIRE

    Ris-Stalpers, C.; Kuiper, G G; Faber, P.W.; SCHWEIKERT, H. U.; van Rooij, H C; Zegers, N.D.; Hodgins, M B; Degenhart, H J; Trapman, J; Brinkmann, A.O.

    1990-01-01

    Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symptoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. We report a G----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 a...

  14. Co-option of the piRNA Pathway for Germline-Specific Alternative Splicing of C. elegans TOR

    OpenAIRE

    Sergio Barberán-Soler; Laura Fontrodona; Anna Ribó; Ayelet T. Lamm; Camilla Iannone; Julián Cerón; Ben Lehner; Juan Valcárcel

    2014-01-01

    Many eukaryotic genes contain embedded antisense transcripts and repetitive sequences of unknown function. We report that male germline-specific expression of an antisense transcript contained in an intron of C. elegans Target of Rapamycin (TOR, let-363) is associated with (1) accumulation of endo-small interfering RNAs (siRNAs) against an embedded Helitron transposon and (2) activation of an alternative 3′ splice site of TOR. The germline-specific Argonaute proteins PRG-1 and CSR-1, which pa...

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

    OpenAIRE

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

    1992-01-01

    In the course of defining mutations causing Tay-Sachs disease (TSD) in non-Jewish patients and carriers from the British Isles, we identified a guanine to adenine change (also previously described) in the obligatory GT sequence of the donor splice site at the 5' end of intron 9 of the hexosaminidase alpha peptide gene. Of 24 unrelated mutant chromosomes from 20 non-Jewish subjects (15 TSD carriers, four TSD patients, and one TSD fetus), five had mutations common in the Ashkenazi Jewish commun...

  16. Quality control of cadweld (mechanical) splices

    International Nuclear Information System (INIS)

    Test data for cadweld splicing of reinforcing steel collected during a study of quality assurance practices on nine nuclear power plant construction projects are presented and evaluated. These data lead to an important hypothesis that the visual inspection identifies procedural deficiencies, and the tensile test identifies material defects. It is also suggested that a material testing program and the visual inspection will detect essentially all substandard cadwell splices. This would permit the deletion of the expensive tensile testing program. Accordingly, most quality control programs require overtesting and overdocumentation of cadweld splices; and furthermore, these programs fail to recognize material defects. The project specifications and quality control requirements for the nine projects are compared. Where possible, these are evaluated against the industry standards and Federal regulations. It is shown that there are a number of deficiencies in these standards, and that in most cases, the testing requirements are not commensurate with the quality that is being achieved in the field

  17. Splicing variants of porcine synphilin-1

    DEFF Research Database (Denmark)

    Larsen, Knud Erik; Madsen, Lone Bruhn; Farajzadeh, Leila;

    2015-01-01

    (90%) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing......RNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa) synphilin-1 cDNA (SNCAIP) and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1) of 919 amino acids which shows a high similarity to human...... variants and a novel splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation....

  18. 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...... cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor...

  19. Methylation affects transposition and splicing of a large CACTA transposon from a MYB transcription factor regulating anthocyanin synthase genes in soybean seed coats.

    Directory of Open Access Journals (Sweden)

    Gracia Zabala

    Full Text Available We determined the molecular basis of three soybean lines that vary in seed coat color at the R locus which is thought to encode a MYB transcription factor. RM55-r(m is homozygous for a mutable allele (r(m that specifies black and brown striped seeds; RM30-R* is a stable black revertant isoline derived from the mutable line; and RM38-r has brown seed coats due to a recessive r allele shown to translate a truncated MYB protein. Using long range PCR, 454 sequencing of amplicons, and whole genome re-sequencing, we determined that the variegated RM55-r(m line had a 13 kb CACTA subfamily transposon insertion (designated TgmR* at a position 110 bp from the beginning of Intron2 of the R locus, Glyma09g36983. Although the MYB encoded by R was expressed at only very low levels in older seed coats of the black revertant RM30-R* line, it upregulated expression of anthocyanidin synthase genes (ANS2, ANS3 to promote the synthesis of anthocyanins. Surprisingly, the RM30-R* revertant also carried the 13 kb TgmR* insertion in Intron2. Using RNA-Seq, we showed that intron splicing was accurate, albeit at lower levels, despite the presence of the 13 kb TgmR* element. As determined by whole genome methylation sequencing, we demonstrate that the TgmR* sequence was relatively more methylated in RM30-R* than in the mutable RM55-r(m progenitor line. The stabilized and more methylated RM30-R* revertant line apparently lacks effective binding of a transposae to its subterminal repeats, thus allowing intron splicing to proceed resulting in sufficient MYB protein to stimulate anthocyanin production and thus black seed coats. In this regard, the TgmR* element in soybean resembles McClintock's Spm-suppressible and change-of-state alleles of maize. This comparison explains the opposite effects of the TgmR* element on intron splicing of the MYB gene in which it resides depending on the methylation state of the element.

  20. Intron-exon organization of the active human protein S gene PS. alpha. and its pseudogene PS. beta. : Duplication and silencing during primate evolution

    Energy Technology Data Exchange (ETDEWEB)

    Ploos van Amstel, H.; Reitsma, P.H.; van der Logt, C.P.; Bertina, R.M. (University Hospital, Leiden (Netherlands))

    1990-08-28

    The human protein S locus on chromosome 3 consists of two protein S genes, PS{alpha} and PS{beta}. Here the authors report the cloning and characterization of both genes. Fifteen exons of the PS{alpha} gene were identified that together code for protein S mRNA as derived from the reported protein S cDNAs. Analysis by primer extension of liver protein S mRNA, however, reveals the presence of two mRNA forms that differ in the length of their 5{prime}-noncoding region. Both transcripts contain a 5{prime}-noncoding region longer than found in the protein S cDNAs. The two products may arise from alternative splicing of an additional intron in this region or from the usage of two start sites for transcription. The intron-exon organization of the PS{alpha} gene fully supports the hypothesis that the protein S gene is the product of an evolutional assembling process in which gene modules coding for structural/functional protein units also found in other coagulation proteins have been put upstream of the ancestral gene of a steroid hormone binding protein. The PS{beta} gene is identified as a pseudogene. It contains a large variety of detrimental aberrations, viz., the absence of exon I, a splice site mutation, three stop codons, and a frame shift mutation. Overall the two genes PS{alpha} and PS{beta} show between their exonic sequences 96.5% homology. Southern analysis of primate DNA showed that the duplication of the ancestral protein S gene has occurred after the branching of the orangutan from the African apes. A nonsense mutation that is present in the pseudogene of man also could be identified in one of the two protein S genes of both chimpanzee and gorilla. This implicates that silencing of one of the two protein S genes must have taken place before the divergence of the three African apes.

  1. Structural and functional characterization of ribosomal protein gene introns in sponges.

    Science.gov (United States)

    Perina, Drago; Korolija, Marina; Mikoč, Andreja; Roller, Maša; Pleše, Bruna; Imešek, Mirna; Morrow, Christine; Batel, Renato; Ćetković, Helena

    2012-01-01

    Ribosomal protein genes (RPGs) are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs). These ancient ncRNAs are small nucleolar RNAs (snoRNAs) essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanisms such as intron/exon gain/loss. snoRNAs in basal metazoans are poorly characterized. We examined 449 RPG introns, in total, from four demosponges: Amphimedon queenslandica, Suberites domuncula, Suberites ficus and Suberites pagurorum and showed that RPG introns from A. queenslandica share position conservancy and some structural similarity with "higher" metazoans. Moreover, our study indicates that mobile element insertions play an important role in the evolution of their size. In four sponges 51 snoRNAs were identified. The analysis showed discrepancies between the snoRNA pools of orthologous RPG introns between S. domuncula and A. queenslandica. Furthermore, these two sponges show as much conservancy of RPG intron positions between each other as between themselves and human. Sponges from the Suberites genus show consistency in RPG intron position conservation. However, significant differences in some of the orthologous RPG introns of closely related sponges were observed. This indicates that RPG introns are dynamic even on these shorter evolutionary time scales. PMID:22880015

  2. Structural and functional characterization of ribosomal protein gene introns in sponges.

    Directory of Open Access Journals (Sweden)

    Drago Perina

    Full Text Available Ribosomal protein genes (RPGs are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs. These ancient ncRNAs are small nucleolar RNAs (snoRNAs essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanisms such as intron/exon gain/loss. snoRNAs in basal metazoans are poorly characterized. We examined 449 RPG introns, in total, from four demosponges: Amphimedon queenslandica, Suberites domuncula, Suberites ficus and Suberites pagurorum and showed that RPG introns from A. queenslandica share position conservancy and some structural similarity with "higher" metazoans. Moreover, our study indicates that mobile element insertions play an important role in the evolution of their size. In four sponges 51 snoRNAs were identified. The analysis showed discrepancies between the snoRNA pools of orthologous RPG introns between S. domuncula and A. queenslandica. Furthermore, these two sponges show as much conservancy of RPG intron positions between each other as between themselves and human. Sponges from the Suberites genus show consistency in RPG intron position conservation. However, significant differences in some of the orthologous RPG introns of closely related sponges were observed. This indicates that RPG introns are dynamic even on these shorter evolutionary time scales.

  3. Longer first introns are a general property of eukaryotic gene structure.

    Directory of Open Access Journals (Sweden)

    Keith R Bradnam

    Full Text Available While many properties of eukaryotic gene structure are well characterized, differences in the form and function of introns that occur at different positions within a transcript are less well understood. In particular, the dynamics of intron length variation with respect to intron position has received relatively little attention. This study analyzes all available data on intron lengths in GenBank and finds a significant trend of increased length in first introns throughout a wide range of species. This trend was found to be even stronger when using high-confidence gene annotation data for three model organisms (Arabidopsis thaliana, Caenorhabditis elegans, and Drosophila melanogaster which show that the first intron in the 5' UTR is--on average--significantly longer than all downstream introns within a gene. A partial explanation for increased first intron length in A. thaliana is suggested by the increased frequency of certain motifs that are present in first introns. The phenomenon of longer first introns can potentially be used to improve gene prediction software and also to detect errors in existing gene annotations.

  4. Progress toward therapy with antisense-mediated splicing modulation

    OpenAIRE

    Du, Liutao; Gatti, Richard A.

    2009-01-01

    Antisense oligonucleotides (AO) or antisense RNA can complementarily bind to a target site in pre-mRNA and regulate gene splicing, either to restore gene function by reprogramming gene splicing or to inhibit gene expression by disrupting splicing. These two applications represent novel therapeutic strategies for several types of diseases such as genetic disorders, cancers and infectious diseases. In this review, the recent developments and applications of antisense-mediated splicing modulatio...

  5. Cotranscriptional splicing efficiency differs dramatically between Drosophila and mouse

    OpenAIRE

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

    2012-01-01

    Spliceosome assembly and/or splicing of a nascent transcript may be crucial for proper isoform expression and gene regulation in higher eukaryotes. It has been shown that cotranscriptional splicing occurs efficiently in Drosophila, but there are not comparable genome-wide nascent splicing data from mammals. To provide this comparison, the authors analyzed a recently generated, high-throughput sequencing data set of mouse liver nascent RNA. Cotranscriptional splicing is approximately twofold l...

  6. Alternative splicing of DNA damage response genes and gastrointestinal cancers

    OpenAIRE

    Rahmutulla, Bahityar; Matsushita, Kazuyuki; Nomura, Fumio

    2014-01-01

    Alternative splicing, which is a common phenomenon in mammalian genomes, is a fundamental process of gene regulation and contributes to great protein diversity. Alternative splicing events not only occur in the normal gene regulation process but are also closely related to certain diseases including cancer. In this review, we briefly demonstrate the concept of alternative splicing and DNA damage and describe the association of alternative splicing and cancer pathogenesis, focusing on the pote...

  7. RNA structure and the mechanisms of alternative splicing

    OpenAIRE

    McManus, C. Joel; Graveley, Brenton R.

    2011-01-01

    Alternative splicing is a widespread means of increasing protein diversity and regulating gene expression in eukaryotes. Much progress has been made in understanding the proteins involved in regulating alternative splicing, the sequences they bind to, and how these interactions lead to changes in splicing patterns. However, several recent studies have identified other players involved in regulating alternative splicing. A major theme emerging from these studies is that RNA secondary structure...

  8. Evolution of alternative splicing in primate brain transcriptomes

    OpenAIRE

    Lin, Lan; Shen, Shihao; Jiang, Peng; Sato, Seiko; Davidson, Beverly L.; Xing, Yi

    2010-01-01

    Alternative splicing is a predominant form of gene regulation in higher eukaryotes. The evolution of alternative splicing provides an important mechanism for the acquisition of novel gene functions. In this work, we carried out a genome-wide phylogenetic survey of lineage-specific splicing patterns in the primate brain, via high-density exon junction array profiling of brain transcriptomes of humans, chimpanzees and rhesus macaques. We identified 509 genes showing splicing differences among t...

  9. COL1A1 transgene expression in stably transfected osteoblastic cells. Relative contributions of first intron, 3'-flanking sequences, and sequences derived from the body of the human COL1A1 minigene

    Science.gov (United States)

    Breault, D. T.; Lichtler, A. C.; Rowe, D. W.

    1997-01-01

    Collagen reporter gene constructs have be used to identify cell-specific sequences needed for transcriptional activation. The elements required for endogenous levels of COL1A1 expression, however, have not been elucidated. The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50-100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3'-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-independent 5-fold increase in CAT activity. In contrast the 3'-flanking sequences gave rise to a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.

  10. SpliceMiner: a high-throughput database implementation of the NCBI Evidence Viewer for microarray splice variant analysis

    OpenAIRE

    Liu Hongfang; Ryan Michael C; Kahn Ari B; Zeeberg Barry R; Jamison D Curtis; Weinstein John N

    2007-01-01

    Abstract Background There are many fewer genes in the human genome than there are expressed transcripts. Alternative splicing is the reason. Alternatively spliced transcripts are often specific to tissue type, developmental stage, environmental condition, or disease state. Accurate analysis of microarray expression data and design of new arrays for alternative splicing require assessment of probes at the sequence and exon levels. Description SpliceMiner is a web interface for querying Evidenc...

  11. The epithelial splicing factors ESRP1 and ESRP2 positively and negatively regulate diverse types of alternative splicing events

    OpenAIRE

    Warzecha, Claude C.; Shen, Shihao; Xing, Yi; Carstens, Russ P.

    2009-01-01

    Cell-type and tissue-specific alternative splicing events are regulated by combinatorial control involving both abundant RNA binding proteins as well as those with more discrete expression and specialized functions. Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2) are recently discovered epithelial-specific RNA binding proteins that promote splicing of the epithelial variant of the FGFR2, ENAH, CD44 and CTNND1 transcripts. To catalogue a larger set of splicing events under th...

  12. Absence of the intron-D-exon of c-Ha-ras oncogene in the hermaphroditic fish Rivulus marmoratus (Teleostei: Rivulidae).

    Science.gov (United States)

    Lee, J S; Choe, J; Park, E H

    1994-11-01

    We have cloned ras genes from the hermaphroditic fish Rivulus marmoratus genomic library by screening about 3.0 x 10(5) genomic clones. When one out of 19 ras genomic clones was sequenced, it showed 95.8% amino acid homology to the human c-Ha-ras gene and was named the Rivulus c-Ha-ras gene. The Rivulus c-Ha-ras gene spanned about 3.7 kb and consisted of four exons encoding 189 amino acids. The exon-intron boundaries also coincided with the rule of GT/AG of consensus splice acceptor and donor sequences. The Rivulus c-Ha-ras gene does not, however, have the intron-D-exon (IDX) that has been reported to exist between the third and the fourth exon of the mammalian c-Ha-ras genes, and which is involved in negative control of p21 c-Ha-ras expression and transforming activity of this gene. This is the first report on the structure of the fish c-Ha-ras gene. PMID:7703908

  13. Is “Junk” DNA Mostly Intron DNA?

    OpenAIRE

    Wong, Gane Ka-Shu; Passey, Douglas A.; Huang, Ying-zong; Yang, Zhiyong; Yu, Jun

    2000-01-01

    Among higher eukaryotes, very little of the genome codes for protein. What is in the rest of the genome, or the “junk” DNA, that, in Homo sapiens, is estimated to be almost 97% of the genome? Is it possible that much of this “junk” is intron DNA? This is not a question that can be answered just by looking at the published data, even from the finished genomes. One cannot assume that there are no genes in a sequenced region, just because no genes were annotated. We introduce another approach to...

  14. Genomic organization and splicing evolution of the doublesex gene, a Drosophila regulator of sexual differentiation, in the dengue and yellow fever mosquito Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Arcà Bruno

    2011-02-01

    Full Text Available Abstract Background In the model system Drosophila melanogaster, doublesex (dsx is the double-switch gene at the bottom of the somatic sex determination cascade that determines the differentiation of sexually dimorphic traits. Homologues of dsx are functionally conserved in various dipteran species, including the malaria vector Anopheles gambiae. They show a striking conservation of sex-specific regulation, based on alternative splicing, and of the encoded sex-specific proteins, which are transcriptional regulators of downstream terminal genes that influence sexual differentiation of cells, tissues and organs. Results In this work, we report on the molecular characterization of the dsx homologue in the dengue and yellow fever vector Aedes aegypti (Aeadsx. Aeadsx produces sex-specific transcripts by alternative splicing, which encode isoforms with a high degree of identity to Anopheles gambiae and Drosophila melanogaster homologues. Interestingly, Aeadsx produces an additional novel female-specific splicing variant. Genomic comparative analyses between the Aedes and Anopheles dsx genes revealed a partial conservation of the exon organization and extensive divergence in the intron lengths. An expression analysis showed that Aeadsx transcripts were present from early stages of development and that sex-specific regulation starts at least from late larval stages. The analysis of the female-specific untranslated region (UTR led to the identification of putative regulatory cis-elements potentially involved in the sex-specific splicing regulation. The Aedes dsx sex-specific splicing regulation seems to be more complex with the respect of other dipteran species, suggesting slightly novel evolutionary trajectories for its regulation and hence for the recruitment of upstream splicing regulators. Conclusions This study led to uncover the molecular evolution of Aedes aegypti dsx splicing regulation with the respect of the more closely related Culicidae

  15. Auxiliary splice factor U2AF26 and transcription factor Gfi1 cooperate directly in regulating CD45 alternative splicing.

    NARCIS (Netherlands)

    Heyd, F.; Dam, G.B. ten; Moroy, T.

    2006-01-01

    By alternative splicing, different isoforms of the transmembrane tyrosine phosphatase CD45 are generated that either enhance or limit T cell receptor signaling. We report here that CD45 alternative splicing is regulated by cooperative action of the splice factor U2AF26 and the transcription factor G

  16. Evolutionary Dynamics of Intron Size, Genome Size, and Physiological Correlates in Archosaurs

    OpenAIRE

    Waltari, Eric; Edwards, Scott

    2002-01-01

    It has been proposed that intron and genome sizes in birds are reduced in comparison with mammals because of the metabolic demands of flight. To test this hypothesis, we examined the sizes of 14 introns in a nonflying relative of birds, the American alligator (Alligator mississippiensis), and in 19 flighted and flightless birds in 12 taxonomic orders. Our results indicate that a substantial fraction (66%) of the reduction in intron size as well as in genome size had already occurred in nonfly...

  17. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    DEFF Research Database (Denmark)

    de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida;

    2014-01-01

    Rare sequence variants in "high-risk" disease genes, often referred as unclassified variants (UVs), pose a serious challenge to genetic testing. However, UVs resulting in splicing alterations can be readily assessed by in vitro assays. Unfortunately, analytical and clinical interpretation of thes...

  18. Alternative-splicing-mediated gene expression

    Science.gov (United States)

    Wang, Qianliang; Zhou, Tianshou

    2014-01-01

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

  19. Approaches to link RNA secondary structures with splicing regulation

    DEFF Research Database (Denmark)

    Plass, Mireya; Eyras, Eduardo

    2014-01-01

    facilitating or hindering the interaction with factors and small nuclear ribonucleoproteins (snRNPs) that regulate splicing. Moreover, the secondary structure could play a fundamental role in the splicing of yeast species, which lack many of the regulatory splicing factors present in metazoans. This chapter......In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either by...

  20. What Can Domesticated Genes Tell Us about the Intron Gain in Mammals?

    Directory of Open Access Journals (Sweden)

    Dušan Kordiš

    2012-01-01

    Full Text Available Domesticated genes, originating from retroelements or from DNA-transposons, constitute an ideal system for testing the hypothesis on the absence of intron gain in mammals. Since single-copy domesticated genes originated from the intronless multicopy transposable elements, the ancestral intron state for domesticated genes is zero. A phylogenomic approach has been used to analyse all domesticated genes in mammals and chordates that originated from the coding parts of transposable elements. A significant amount of intron gain was found only in domesticated genes of placental mammals, where more than 70 cases were identified. De novo gained introns show clear positional bias, since they are distributed mainly in 5′ UTR and coding regions, while 3′ UTR introns are very rare. In the coding regions of some domesticated genes up to 8 de novo gained introns have been found. Surprisingly, the majority of intron gains have occurred in the ancestor of placental mammals. Domesticated genes could constitute an excellent system on which to analyse the mechanisms of intron gain. This paper summarizes the current understanding of intron gain in mammals.

  1. Using intron position conservation for homology-based gene prediction.

    Science.gov (United States)

    Keilwagen, Jens; Wenk, Michael; Erickson, Jessica L; Schattat, Martin H; Grau, Jan; Hartung, Frank

    2016-05-19

    Annotation of protein-coding genes is very important in bioinformatics and biology and has a decisive influence on many downstream analyses. Homology-based gene prediction programs allow for transferring knowledge about protein-coding genes from an annotated organism to an organism of interest.Here, we present a homology-based gene prediction program called GeMoMa. GeMoMa utilizes the conservation of intron positions within genes to predict related genes in other organisms. We assess the performance of GeMoMa and compare it with state-of-the-art competitors on plant and animal genomes using an extended best reciprocal hit approach. We find that GeMoMa often makes more precise predictions than its competitors yielding a substantially increased number of correct transcripts. Subsequently, we exemplarily validate GeMoMa predictions using Sanger sequencing. Finally, we use RNA-seq data to compare the predictions of homology-based gene prediction programs, and find again that GeMoMa performs well.Hence, we conclude that exploiting intron position conservation improves homology-based gene prediction, and we make GeMoMa freely available as command-line tool and Galaxy integration. PMID:26893356

  2. Single Mode Fiber Optic Connectors And Splices

    Science.gov (United States)

    Woods, John G.

    1984-08-01

    There is a trend toward increasing use of single mode transmission, particularly in telecommunications where high data bit rates are transmitted for long distances. Inter-connections of multimode fibers can be made in a number of ways, using ferrules, v-grooves, elastomeric splices, etc. However, the connection of single mode fibers, which have core diameters of 4 to 13 μm, requires more precise alignment than do the multimode fibers having core diameters of 50 μm or more. At TRW, we have adapted the four rod alignment guide concept for single mode fiber inter-connections. The principle of this OPTAGUIDE* alignment guide is presented. The single mode connectors and splices use the four rod scheme with an index matching material to eliminate or reduce the losses incurred through fiber end roughness or angularity. We are able to produce demountable connectors for 80/4.4 pm fibers having typical insertion losses of 1.0dB. The main factors in obtaining this result are the naturally precise fiber alignment provided by the alignment guide, and the ability of several manufacturers to maintain tight diametral and core offset tolerances. The single mode OPTALIGN* SM Connectors have been subjected to performance and environmental tests including repeated matings, temperature cycle and vibration. The results of these tests are described in this paper. A feature of the OPTALIGN* SM Connectors is the relative ease and speed of attachment to fiber optic cable in the field, without the use of epoxy or polishing procedures. The alignment guide concept has also been applied to permanent single mode splices. The splicing procedure is simple to perform in the field without expensive or delicate equipment. Construction and assembly procedures of the demountable connectors and permanent splices will be described with the aid of diagrams and photographs.

  3. An ancient spliceosomal intron in the ribosomal protein L7a gene (Rpl7a of Giardia lamblia

    Directory of Open Access Journals (Sweden)

    Gray Michael W

    2005-08-01

    Full Text Available Abstract Background Only one spliceosomal-type intron has previously been identified in the unicellular eukaryotic parasite, Giardia lamblia (a diplomonad. This intron is only 35 nucleotides in length and is unusual in possessing a non-canonical 5' intron boundary sequence, CT, instead of GT. Results We have identified a second spliceosomal-type intron in G. lamblia, in the ribosomal protein L7a gene (Rpl7a, that possesses a canonical GT 5' intron boundary sequence. A comparison of the two known Giardia intron sequences revealed extensive nucleotide identity at both the 5' and 3' intron boundaries, similar to the conserved sequence motifs recently identified at the boundaries of spliceosomal-type introns in Trichomonas vaginalis (a parabasalid. Based on these observations, we searched the partial G. lamblia genome sequence for these conserved features and identified a third spliceosomal intron, in an unassigned open reading frame. Our comprehensive analysis of the Rpl7a intron in other eukaryotic taxa demonstrates that it is evolutionarily conserved and is an ancient eukaryotic intron. Conclusion An analysis of the phylogenetic distribution and properties of the Rpl7a intron suggests its utility as a phylogenetic marker to evaluate particular eukaryotic groupings. Additionally, analysis of the G. lamblia introns has provided further insight into some of the conserved and unique features possessed by the recently identified spliceosomal introns in related organisms such as T. vaginalis and Carpediemonas membranifera.

  4. Discovery of a mammalian splice variant of myostatin that stimulates myogenesis.

    Directory of Open Access Journals (Sweden)

    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

  5. Contrasting evolutionary histories of two introns of the duchenne muscular dystrophy gene, Dmd, in humans.

    Science.gov (United States)

    Nachman, M W; Crowell, S L

    2000-08-01

    The Duchenne muscular dystrophy (Dmd) locus lies in a region of the X chromosome that experiences a high rate of recombination and is thus expected to be relatively unaffected by the effects of selection on nearby genes. To provide a picture of nucleotide variability at a high-recombination locus in humans, we sequenced 5. 4 kb from two introns of Dmd in a worldwide sample of 41 alleles from Africa, Asia, Europe, and the Americas. These same regions were also sequenced in one common chimpanzee and one orangutan. Dramatically different patterns of genetic variation were observed at these two introns, which are separated by >500 kb of DNA. Nucleotide diversity at intron 44 pi = 0.141% was more than four times higher than nucleotide diversity at intron 7 pi = 0.034% despite similar levels of divergence for these two regions. Intron 7 exhibited significant linkage disequilibrium extending over 10 kb and also showed a significant excess of rare polymorphisms. In contrast, intron 44 exhibited little linkage disequilibrium and no skew in the frequency distribution of segregating sites. Intron 7 was much more variable in Africa than in other continents, while intron 44 displayed similar levels of variability in different geographic regions. Comparison of intraspecific polymorphism to interspecific divergence using the HKA test revealed a significant reduction in variability at intron 7 relative to intron 44, and this effect was most pronounced in the non-African samples. These results are best explained by positive directional selection acting at or near intron 7 and demonstrate that even genes in regions of high recombination may be influenced by selection at linked sites. PMID:10924480

  6. Intron Invasions Trace Algal Speciation and Reveal Nearly Identical Arctic and Antarctic Micromonas Populations.

    Science.gov (United States)

    Simmons, Melinda P; Bachy, Charles; Sudek, Sebastian; van Baren, Marijke J; Sudek, Lisa; Ares, Manuel; Worden, Alexandra Z

    2015-09-01

    Spliceosomal introns are a hallmark of eukaryotic genes that are hypothesized to play important roles in genome evolution but have poorly understood origins. Although most introns lack sequence homology to each other, new families of spliceosomal introns that are repeated hundreds of times in individual genomes have recently been discovered in a few organisms. The prevalence and conservation of these introner elements (IEs) or introner-like elements in other taxa, as well as their evolutionary relationships to regular spliceosomal introns, are still unknown. Here, we systematically investigate introns in the widespread marine green alga Micromonas and report new families of IEs, numerous intron presence-absence polymorphisms, and potential intron insertion hot-spots. The new families enabled identification of conserved IE secondary structure features and establishment of a novel general model for repetitive intron proliferation across genomes. Despite shared secondary structure, the IE families from each Micromonas lineage bear no obvious sequence similarity to those in the other lineages, suggesting that their appearance is intimately linked with the process of speciation. Two of the new IE families come from an Arctic culture (Micromonas Clade E2) isolated from a polar region where abundance of this alga is increasing due to climate induced changes. The same two families were detected in metagenomic data from Antarctica--a system where Micromonas has never before been reported. Strikingly high identity between the Arctic isolate and Antarctic coding sequences that flank the IEs suggests connectivity between populations in the two polar systems that we postulate occurs through deep-sea currents. Recovery of Clade E2 sequences in North Atlantic Deep Waters beneath the Gulf Stream supports this hypothesis. Our research illuminates the dynamic relationships between an unusual class of repetitive introns, genome evolution, speciation, and global distribution of this

  7. Compound heterozygous DUOX2 gene mutations (c.2335-1G>C/c.3264_3267delCAGC) associated with congenital hypothyroidism. Characterization of complex cryptic splice sites by minigene analysis.

    Science.gov (United States)

    Belforte, Fiorella S; Citterio, Cintia E; Testa, Graciela; Olcese, María Cecilia; Sobrero, Gabriela; Miras, Mirta B; Targovnik, Héctor M; Rivolta, Carina M

    2016-01-01

    Iodide Organification defects (IOD) represent 10% of cases of congenital hypothyroidism (CH) being the main genes affected that of TPO (thyroid peroxidase) and DUOX2 (dual oxidasa 2). From a patient with clinical and biochemical criteria suggestive with CH associated with IOD, TPO and DUOX2 genes were analyzed by means of PCR-Single Strand Conformation Polymorphism analysis and sequencing. A novel heterozygous compound to the mutations c.2335-1G>C (paternal mutation, intron 17) and c.3264_3267delCAGC (maternal mutation, exon 24) was identified in the DUOX2 gene. Ex-vivo splicing assays and subsequent RT-PCR and sequencing analyses were performed on mRNA isolated from the HeLa cells transfected with wild-type and mutant pSPL3 expression vectors. The wild-type and c.2335-1G>C mutant alleles result in the complete inclusion or exclusion of exon 18, or in the activation of an exonic cryptic 5' ss with the consequent deletion of 169 bp at the end of this exon. However, we observed only a band of the expected size in normal thyroid tissue by RT-PCR. Additionally, the c.2335-1G>C mutation activates an unusual cryptic donor splice site in intron 17, located at position -14 of the authentic intron 17/exon 18 junction site, with an insertion of the last 14 nucleotides of the intron 17 in mutant transcripts with complete and partial inclusion of exon 18. The theoretical consequences of splice site mutation, predicted with the bioinformatics NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses confirm that c.2335-1G>C mutant allele would result in the abolition of the authentic splice acceptor site. The results suggest the coexistence in our patient of four putative truncated proteins of 786, 805, 806 and 1105 amino acids, with conservation of peroxidase-like domain and loss of gp91(phox)/NOX2-like domain. In conclusion a novel heterozygous compound was identified being responsible of

  8. SpliceMiner: a high-throughput database implementation of the NCBI Evidence Viewer for microarray splice variant analysis

    Directory of Open Access Journals (Sweden)

    Liu Hongfang

    2007-03-01

    Full Text Available Abstract Background There are many fewer genes in the human genome than there are expressed transcripts. Alternative splicing is the reason. Alternatively spliced transcripts are often specific to tissue type, developmental stage, environmental condition, or disease state. Accurate analysis of microarray expression data and design of new arrays for alternative splicing require assessment of probes at the sequence and exon levels. Description SpliceMiner is a web interface for querying Evidence Viewer Database (EVDB. EVDB is a comprehensive, non-redundant compendium of splice variant data for human genes. We constructed EVDB as a queryable implementation of the NCBI Evidence Viewer (EV. EVDB is based on data obtained from NCBI Entrez Gene and EV. The automated EVDB build process uses only complete coding sequences, which may or may not include partial or complete 5' and 3' UTRs, and filters redundant splice variants. Unlike EV, which supports only one-at-a-time queries, SpliceMiner supports high-throughput batch queries and provides results in an easily parsable format. SpliceMiner maps probes to splice variants, effectively delineating the variants identified by a probe. Conclusion EVDB can be queried by gene symbol, genomic coordinates, or probe sequence via a user-friendly web-based tool we call SpliceMiner (http://discover.nci.nih.gov/spliceminer. The EVDB/SpliceMiner combination provides an interface with human splice variant information and, going beyond the very valuable NCBI Evidence Viewer, supports fluent, high-throughput analysis. Integration of EVDB information into microarray analysis and design pipelines has the potential to improve the analysis and bioinformatic interpretation of gene expression data, for both batch and interactive processing. For example, whenever a gene expression value is recognized as important or appears anomalous in a microarray experiment, the interactive mode of SpliceMiner can be used quickly and easily to

  9. In trangenic rice, alpha- and beta-tubulin regulatory sequences control GUS amount and distribution through intron mediated enhancement and intron dependent spatial expression.

    Science.gov (United States)

    Gianì, Silvia; Altana, Andrea; Campanoni, Prisca; Morello, Laura; Breviario, Diego

    2009-04-01

    The genomic upstream sequence of the rice tubulin gene OsTub6 has been cloned, sequenced and characterized. The 5'UTR sequence is interrupted by a 446 bp long leader intron. This feature is shared with two other rice beta-tubulin genes (OsTub4 and OsTub1) that, together with OsTub6, group in the same clade in the evolutionary phylogenetic tree of plant beta-tubulins. Similarly to OsTub4, the leader intron of OsTub6 is capable of sustaining intron mediated enhancement (IME) of gene expression, in transient expression assays. A general picture is drawn for three rice alpha-tubulin and two rice beta-tubulin genes in which the first intron of the coding sequence for the formers and the intron present in the 5'UTR for the latters, are important elements for controlling gene expression. We used OsTua2:GUS, OsTua3:GUS, OsTub4:GUS and OsTub6:GUS chimeric constructs to investigate the in vivo pattern of beta-glucuronidase (GUS) expression in transgenic rice plants. The influence of the regulatory introns on expression patterns was evaluated for two of them, OsTua2 and OsTub4. We have thus characterized distinct patterns of expression attributable to each tubulin isotype and we have shown that the presence of the regulatory intron can greatly influence both the amount and the actual site of expression. We propose the term Intron Dependent Spatial Expression (IDSE) to highlight this latter effect. PMID:18668337

  10. 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.

  11. NOVEL SPLICING MUTATION OF COL1A1 GENE CAUSING OSTEOGENESIS IMPERFECTA TYPE I IN CHINESE PEDIGREE

    Institute of Scientific and Technical Information of China (English)

    WU Xiao-lin; GU Ming-min; CUI Bing; LI Xi-hua; LU Zhen-yu; WANG Zhu-gang; YUAN Wen-tao; SONG Huai-dong

    2007-01-01

    Objective To detect the peculiar mutation in a Chinese family with osteogenesis imperfecta,COL1A1 and COL1A2 being analysed. Methods A genome screen was undertaken covering COL1A1 at 17q21-22 and COL1A2 at 7q22.1. The Linkage ( Version 5.1 ) was used for 2-point analysis. DNA sequencing was used to screen and identify the mutation. Results A linkage to the markers on chromosome 17q21-22 was observed. Sequence analysis of COL1A1 revealed a splicing mutation ( IVS8-2A > G) that converted the 3' end of intron 8 from AG to GG. Conclusion This mutation ( IVS 8-2A > G) is novel, and has not yet been registered in the Human Type Ⅰ and Type Ⅲ Collagen Mutations Database.

  12. Conserved functional antagonism of CELF and MBNL proteins controls stem cell-specific alternative splicing in planarians.

    Science.gov (United States)

    Solana, Jordi; Irimia, Manuel; Ayoub, Salah; Orejuela, Marta Rodriguez; Zywitza, Vera; Jens, Marvin; Tapial, Javier; Ray, Debashish; Morris, Quaid; Hughes, Timothy R; Blencowe, Benjamin J; Rajewsky, Nikolaus

    2016-01-01

    In contrast to transcriptional regulation, the function of alternative splicing (AS) in stem cells is poorly understood. In mammals, MBNL proteins negatively regulate an exon program specific of embryonic stem cells; however, little is known about the in vivo significance of this regulation. We studied AS in a powerful in vivo model for stem cell biology, the planarian Schmidtea mediterranea. We discover a conserved AS program comprising hundreds of alternative exons, microexons and introns that is differentially regulated in planarian stem cells, and comprehensively identify its regulators. We show that functional antagonism between CELF and MBNL factors directly controls stem cell-specific AS in planarians, placing the origin of this regulatory mechanism at the base of Bilaterians. Knockdown of CELF or MBNL factors lead to abnormal regenerative capacities by affecting self-renewal and differentiation sets of genes, respectively. These results highlight the importance of AS interactions in stem cell regulation across metazoans. PMID:27502555

  13. Splicing therapy for neuromuscular disease ☆

    OpenAIRE

    Andrew G. L. Douglas; Wood, Matthew J. A.

    2013-01-01

    Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) are two of the most common inherited neuromuscular diseases in humans. Both conditions are fatal and no clinically available treatments are able to significantly alter disease course in either case. However, by manipulation of pre-mRNA splicing using antisense oligonucleotides, defective transcripts from the DMD gene and from the SMN2 gene in SMA can be modified to once again produce protein and restore function. A large numb...

  14. Resolving deconvolution ambiguity in gene alternative splicing

    Directory of Open Access Journals (Sweden)

    Hubbell Earl

    2009-08-01

    Full Text Available Abstract Background For many gene structures it is impossible to resolve intensity data uniquely to establish abundances of splice variants. This was empirically noted by Wang et al. in which it was called a "degeneracy problem". The ambiguity results from an ill-posed problem where additional information is needed in order to obtain an unique answer in splice variant deconvolution. Results In this paper, we analyze the situations under which the problem occurs and perform a rigorous mathematical study which gives necessary and sufficient conditions on how many and what type of constraints are needed to resolve all ambiguity. This analysis is generally applicable to matrix models of splice variants. We explore the proposal that probe sequence information may provide sufficient additional constraints to resolve real-world instances. However, probe behavior cannot be predicted with sufficient accuracy by any existing probe sequence model, and so we present a Bayesian framework for estimating variant abundances by incorporating the prediction uncertainty from the micro-model of probe responsiveness into the macro-model of probe intensities. Conclusion The matrix analysis of constraints provides a tool for detecting real-world instances in which additional constraints may be necessary to resolve splice variants. While purely mathematical constraints can be stated without error, real-world constraints may themselves be poorly resolved. Our Bayesian framework provides a generic solution to the problem of uniquely estimating transcript abundances given additional constraints that themselves may be uncertain, such as regression fit to probe sequence models. We demonstrate the efficacy of it by extensive simulations as well as various biological data.

  15. Alternative Splicing Programs in Prostate Cancer

    OpenAIRE

    Claudio Sette

    2013-01-01

    Prostate cancer (PCa) remains one of the most frequent causes of death for cancer in the male population. Although the initial antiandrogenic therapies are efficacious, PCa often evolves into a hormone-resistant, incurable disease. The genetic and phenotypic heterogeneity of this type of cancer renders its diagnosis and cure particularly challenging. Mounting evidence indicates that alternative splicing, the process that allows production of multiple mRNA variants from each gene, contributes ...

  16. Splicing variants of porcine synphilin-1

    Directory of Open Access Journals (Sweden)

    Knud Larsen

    2015-09-01

    Full Text Available Parkinson's disease (PD, idiopathic and familial, is characterized by degradation of dopaminergic neurons and the presence of Lewy bodies (LB in the substantia nigra. LBs contain aggregated proteins of which α-synuclein is the major component. The protein synphilin-1 interacts and colocalizes with α-synuclein in LBs. The aim of this study was to isolate and characterize porcine synphilin-1 and isoforms hereof with the future perspective to use the pig as a model for Parkinson's disease. The porcine SNCAIP cDNA was cloned by reverse transcriptase PCR. The spatial expression of SNCAIP mRNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa synphilin-1 cDNA (SNCAIP and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1 of 919 amino acids which shows a high similarity to human (90% and to mouse (84% synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation.

  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. A study of alternative splicing in the pig

    Directory of Open Access Journals (Sweden)

    Jørgensen Claus B

    2010-05-01

    Full Text Available Abstract 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 alternative isoforms. With the EST resource for the domestic pig now containing more than one million porcine ESTs, it is possible to identify alternative splice forms of the individual transcripts in this species from the EST data with some confidence. Results The pig EST data generated by the Sino-Danish Pig Genome project has been assembled with publicly available ESTs and made available in the PigEST database. Using the Distiller package 2,515 EST clusters with candidate alternative isoforms were identified in the EST data with high confidence. In agreement with general observations in human 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 transcripts with expression patterns matching those of the EST data. The remaining four genes had tissue-restricted expression of alternative spliced transcripts. Five out of the 16 splice events that were experimentally verified were found to be putative pig specific. Conclusions In accordance with human and rodent studies we estimate that approximately 30% of the porcine genes undergo alternative splicing. We found a good correlation between EST predicted tissue

  19. An amino-terminal variant of the central cannabinoid receptor resulting from alternative splicing.

    Science.gov (United States)

    Shire, D; Carillon, C; Kaghad, M; Calandra, B; Rinaldi-Carmona, M; Le Fur, G; Caput, D; Ferrara, P

    1995-02-24

    The cDNA sequences encoding the central cannabinoid receptor, CB1, are known for two species, rat and human. However, little information concerning the flanking, noncoding regions is presently available. We have isolated two overlapping clones from a human lung cDNA library with CB1 cDNA inserts. One of these, cann7, contains a short stretch of the CB1 coding region and 4 kilobase pairs (kb) of the 3'-untranslated region (UTR), including two polyadenylation signals. The other, cann6, is identical to cann7 upstream from the first polyadenylation signal, and in addition, it contains the whole coding region and extends for 1.8 kb into the 5'-UTR. Comparison of cann6 with the published sequence (Gérard, C. M., Mollereau, C., Vassart, G., and Parmentier, M. (1991) Biochem. J. 279, 129-134) shows the coding regions to be identical, but reveals important differences in the flanking regions. Notably, the cann6 sequence appears to be that of an immature transcript, containing 1.8 kb of an intronic sequence in the 5'-UTR. In addition, polymerase chain reaction amplification of the CB1 coding region in the IM-9 cell line cDNA resulted in two fragments, one containing the whole CB1 coding region and the second lacking a 167-base pair intron within the sequence encoding the amino-terminal tail of the receptor. This alternatively spliced form would translate to an NH2-terminal modified isoform (CB1A) of the receptor, shorter than CB1 by 61 amino acids. In addition, the first 28 amino acids of the putative truncated receptor are completely different from those of CB1, containing more hydrophobic residues. Rat CB1 mRNA is similarly alternatively spliced. A study of the distribution of the human CB1 and CB1A mRNAs by reverse transcription-polymerase chain reaction analysis showed the presence of both CB1 and CB1A throughout the brain and in all the peripheral tissues examined, with CB1A being present in amounts of up to 20% of CB1. PMID:7876112

  20. 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

  1. 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...... suggested as one explanation for the discrepancy between the number of human genes and functional complexity. Here, we carry out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has...... commonly been suggested, and we demonstrate that many of the potential alternative gene products will have markedly different structure and function from their constitutively spliced counterparts. For the vast majority of these alternative isoforms, little evidence exists to suggest they have a role as...

  2. Identification of a truncated splice variant of IL-18 receptor alpha in the human and rat, with evidence of wider evolutionary conservation

    Directory of Open Access Journals (Sweden)

    Chris S. Booker

    2014-09-01

    Full Text Available Interleukin-18 (IL-18 is a pro-inflammatory cytokine which stimulates activation of the nuclear factor kappa beta (NF-κB pathway via interaction with the IL-18 receptor. The receptor itself is formed from a dimer of two subunits, with the ligand-binding IL-18Rα subunit being encoded by the IL18R1 gene. A splice variant of murine IL18r1, which has been previously described, is formed by transcription of an unspliced intron (forming a ‘type II’ IL18r1 transcript and is predicted to encode a receptor with a truncated intracellular domain lacking the capacity to generate downstream signalling. In order to examine the relevance of this finding to human IL-18 function, we assessed the presence of a homologous transcript by reverse transcription-polymerase chain reaction (RT-PCR in the human and rat as another common laboratory animal. We present evidence for type II IL18R1 transcripts in both species. While the mouse and rat transcripts are predicted to encode a truncated receptor with a novel 5 amino acid C-terminal domain, the human sequence is predicted to encode a truncated protein with a novel 22 amino acid sequence bearing resemblance to the ‘Box 1’ motif of the Toll/interleukin-1 receptor (TIR domain, in a similar fashion to the inhibitory interleukin-1 receptor 2. Given that transcripts from these three species are all formed by inclusion of homologous unspliced intronic regions, an analysis of homologous introns across a wider array of 33 species with available IL18R1 gene records was performed, which suggests similar transcripts may encode truncated type II IL-18Rα subunits in other species. This splice variant may represent a conserved evolutionary mechanism for regulating IL-18 activity.

  3. Tissue-specific splicing factor gene expression signatures

    OpenAIRE

    Grosso, A. R.; Gomes, Anita; Barbosa-Morais, Nuno; Caldeira, Sandra; Thorne, Natalie; Grech, Godfrey; Lindern, Marieke; Carmo-Fonseca, Maria

    2008-01-01

    textabstractThe alternative splicing code that controls and coordinates the transcriptome in complex multicellular organisms remains poorly understood. It has long been argued that regulation of alternative splicing relies on combinatorial interactions between multiple proteins, and that tissue-specific splicing decisions most likely result from differences in the concentration and/or activity of these proteins. However, large-scale data to systematically address this issue have just recently...

  4. Alternative Splicing and Its Impact as a Cancer Diagnostic Marker

    OpenAIRE

    Kim, Yun-Ji; Kim, Heui-Soo

    2012-01-01

    Most genes are processed by alternative splicing for gene expression, resulting in the complexity of the transcriptome in eukaryotes. It allows a limited number of genes to encode various proteins with intricate functions. Alternative splicing is regulated by genetic mutations in cis-regulatory factors and epigenetic events. Furthermore, splicing events occur differently according to cell type, developmental stage, and various diseases, including cancer. Genome instability and flexible proteo...

  5. Pre-mRNA splicing in disease and therapeutics

    OpenAIRE

    Singh, Ravi K.; Cooper, Thomas A.

    2012-01-01

    In metazoans, alternative splicing of genes is essential for regulating gene expression and contributing to functional complexity. Computational predictions, comparative genomics, and transcriptome profiling of normal and diseased tissues indicate an unexpectedly high fraction of diseases are caused by mutations that alter splicing. Mutations in cis elements cause mis-splicing of genes that alter gene function and contribute to disease pathology. Mutations of core spliceosomal factors are ass...

  6. Conservation of RNA secondary structures in two intron families including mitochondrial-, chloroplast- and nuclear-encoded members.

    OpenAIRE

    Michel, F; Dujon, B

    1983-01-01

    Two families of fungal mitochondrial introns that include all known sequences have been recognized. These families are now extended to incorporate a plant mitochondrial intron and several introns in chloroplast- and nuclear-encoded rRNA and tRNA precursors. Members of the same family share distinctive sequence stretches and a number of potential RNA secondary structures that would bring these stretches and the intron-exon junctions into relatively close proximity. Using several of these intro...

  7. Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing

    International Nuclear Information System (INIS)

    Adenosine deaminase deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, the authors synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNA showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These change do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency

  8. Splicing mutation in Sbf1 causes nonsyndromic male infertility in the rat.

    Science.gov (United States)

    Liška, František; Chylíková, Blanka; Janků, Michaela; Šeda, Ondřej; Vernerová, Zdeňka; Pravenec, Michal; Křen, Vladimír

    2016-09-01

    In the inbred SHR/OlaIpcv rat colony, we identified males with small testicles and inability to reproduce. By selectively breeding their parents, we revealed the infertility to segregate as an autosomal recessive Mendelian character. No other phenotype was observed in males, and females were completely normal. By linkage using a backcross with Brown Norway strain, we mapped the locus to a 1.2Mbp segment on chromosome 7, harboring 35 genes. Sequencing of candidate genes revealed a G to A substitution in a canonical 'AG' splice site of intron 37 in Sbf1 (SET binding factor 1, alias myotubularin-related protein 5). This leads to either skipping exon 38 or shifting splicing one base downstream, invariantly resulting in frameshift, premature stop codon and truncation of the protein. Western blotting using two anti-Sbf1 antibodies revealed absence of the full-length protein in the mutant testis. Testicles of the mutant males were significantly smaller compared with SHR from 4weeks, peaked at 84% wild-type weight at 6weeks and declined afterward to 28%, reflecting massive germ cell loss. Histological examination revealed lower germ cell number; latest observed germ cell stage were round spermatids, resulting in the absence of sperm in the epididymis (azoospermia). SBF1 is a member of a phosphatase family lacking the catalytical activity. It probably modulates the activity of a phosphoinositol phosphatase MTMR2. Human homozygotes or compound heterozygotes for missense SBF1 mutations exhibit Charcot-Marie-Tooth disease (manifested mainly as progressive neuropathy), while a single mouse knockout reported in the literature identified male infertility as the only phenotype manifestation. PMID:27335132

  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. 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.

  11. Functional roles of alternative splicing factors in human disease.

    Science.gov (United States)

    Cieply, Benjamin; Carstens, Russ P

    2015-01-01

    Alternative splicing (AS) is an important mechanism used to generate greater transcriptomic and proteomic diversity from a finite genome. Nearly all human gene transcripts are alternatively spliced and can produce protein isoforms with divergent and even antagonistic properties that impact cell functions. Many AS events are tightly regulated in a cell-type or tissue-specific manner, and at different developmental stages. AS is regulated by RNA-binding proteins, including cell- or tissue-specific splicing factors. In the past few years, technological advances have defined genome-wide programs of AS regulated by increasing numbers of splicing factors. These splicing regulatory networks (SRNs) consist of transcripts that encode proteins that function in coordinated and related processes that impact the development and phenotypes of different cell types. As such, it is increasingly recognized that disruption of normal programs of splicing regulated by different splicing factors can lead to human diseases. We will summarize examples of diseases in which altered expression or function of splicing regulatory proteins has been implicated in human disease pathophysiology. As the role of AS continues to be unveiled in human disease and disease risk, it is hoped that further investigations into the functions of numerous splicing factors and their regulated targets will enable the development of novel therapies that are directed at specific AS events as well as the biological pathways they impact. PMID:25630614

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

    OpenAIRE

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

    2007-01-01

    Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additi...

  13. Intein-mediated purification of cytotoxic endonuclease I-TevI by insertional inactivation and pH-controllable splicing.

    Science.gov (United States)

    Wu, Wei; Wood, David W; Belfort, Georges; Derbyshire, Victoria; Belfort, Marlene

    2002-11-15

    An intein-mediated approach was developed for expression and affinity purification of a protein that is lethal to Escherichia coli. The protein, I-TevI, is an intron-encoded endonuclease. The approach involved the insertional inactivation of I-TevI with a controllable mini-intein placed in front of a cysteine required for splicing (an I-TevI::intein fusion). The purification was facilitated by a chitin-binding domain inserted into the mini-intein. Affinity purification of the I-TevI::intein fusion precursor on a chitin column was followed by pH-controllable splicing to restore the structure and function of I-TevI. To study the impact of the insertion context on I-TevI inactivation, the chimeric intein was inserted independently in front of seven cysteines of I-TevI. One of the seven intein integrants yielded I-TevI of high activity. This technique is, in principle, generalizable to the expression and purification of other cytotoxic proteins and is amenable to scale-up. PMID:12433989

  14. Ancient, highly polymorphic human major histocompatibility complex DQA1 intron sequence

    Energy Technology Data Exchange (ETDEWEB)

    McGinnis, M.D.; Quinn, D.L.; Lebo, R.V. [Univ. of California, San Francisco, CA (United States); Simons, M.J. [GeneType Pty. Ltd., Fitzroy, Victoria (Australia)

    1994-10-01

    A 438 basepair intron 1 sequence adjacent to exon 2 in the human major histocompatibility complex DQA1 gene defined 16 allelic variants in 69 individuals from wide ethnic backgrounds. In contrast, the most variable coding region spanned by the 247 basepair exon 2 defined 11 allelic variants. Our phylogenetic human intron 1 tree derived by the Bootstrap algorithm reflects the same relative allelic relationships as the reported DQA1 exon 2 have cosegregated since divergence of the human races. Comparison of human alleles to a Rhesus monkey DQA1 first intron sequence found only 10 nucleotide substitutions unique to Rhesus, with the other 428 positions (98%) found in at least one human allele. This high degree of homology reflects the evolutionary stability of intron sequences since these two species diverged over 20 million years ago. Because more intron 1 alleles exist than exon 2 alleles, these polymorphic introns can be used to improve tissue typing for transplantation, paternity testing, and forensics and to derive more complete phylogenetic trees. These results suggest that introns represent a previously underutilized polymorphic resource. 42 refs., 3 figs., 1 tab.

  15. AtRTD2: A Reference Transcript Dataset for accurate quantification of alternative splicing and expression changes in Arabidopsis thaliana RNA-seq data

    KAUST Repository

    Zhang, Runxuan

    2016-05-06

    Background Alternative splicing is the major post-transcriptional mechanism by which gene expression is regulated and affects a wide range of processes and responses in most eukaryotic organisms. RNA-sequencing (RNA-seq) can generate genome-wide quantification of individual transcript isoforms to identify changes in expression and alternative splicing. RNA-seq is an essential modern tool but its ability to accurately quantify transcript isoforms depends on the diversity, completeness and quality of the transcript information. Results We have developed a new Reference Transcript Dataset for Arabidopsis (AtRTD2) for RNA-seq analysis containing over 82k non-redundant transcripts, whereby 74,194 transcripts originate from 27,667 protein-coding genes. A total of 13,524 protein-coding genes have at least one alternatively spliced transcript in AtRTD2 such that about 60% of the 22,453 protein-coding, intron-containing genes in Arabidopsis undergo alternative splicing. More than 600 putative U12 introns were identified in more than 2,000 transcripts. AtRTD2 was generated from transcript assemblies of ca. 8.5 billion pairs of reads from 285 RNA-seq data sets obtained from 129 RNA-seq libraries and merged along with the previous version, AtRTD, and Araport11 transcript assemblies. AtRTD2 increases the diversity of transcripts and through application of stringent filters represents the most extensive and accurate transcript collection for Arabidopsis to date. We have demonstrated a generally good correlation of alternative splicing ratios from RNA-seq data analysed by Salmon and experimental data from high resolution RT-PCR. However, we have observed inaccurate quantification of transcript isoforms for genes with multiple transcripts which have variation in the lengths of their UTRs. This variation is not effectively corrected in RNA-seq analysis programmes and will therefore impact RNA-seq analyses generally. To address this, we have tested different genome

  16. Identification and characterization of a constitutively expressed Ctenopharyngodon idella ADAR1 splicing isoform (CiADAR1a).

    Science.gov (United States)

    Liu, Xiancheng; Huang, Keyi; Hou, Qunhao; Sun, Zhicheng; Wang, Binhua; Lin, Gang; Li, Dongming; Liu, Yong; Xu, Xiaowen; Hu, Chengyu

    2016-10-01

    As one member of ADAR family, ADAR1 (adenosine deaminase acting on RNA 1) can convert adenosine to inosine within dsRNA. There are many ADAR1 splicing isoforms in mammals, including an interferon (IFN) inducible ∼150 kD protein (ADAR1-p150) and a constitutively expressed ∼110 kD protein (ADAR1-p110). The structural diversity of ADAR1 splicing isoforms may reflect their multiple functions. ADAR1 splicing isoforms were also found in fish. In our previous study, we have cloned and identified two different grass carp ADAR1 splicing isoforms, i.e. CiADAR1 and CiADAR1-like, both of them are IFN-inducible proteins. In this paper, we identified a novel CiADAR1 splicing isoform gene (named CiADAR1a). CiADAR1a gene contains 15 exons and 14 introns. Its full-length cDNA is comprised of a 5' UTR (359 bp), a 3' UTR (229 bp) and a 2952 bp ORF encoding a polypeptide of 983 amino acids with one Z-DNA binding domain, three dsRNA binding motifs and a highly conserved hydrolytic deamination domain. CiADAR1a was constitutively expressed in Ctenopharyngodon idella kidney (CIK) cells regardless of Poly I:C stimulation by Western blot assay. In normal condition, CiADAR1a was found to be present mainly in the nucleus. After treatment with Poly I:C, it gradually shifted to cytoplasm. To further investigate the mechanism of transcriptional regulation of CiADAR1a, we cloned and identified its promoter sequence. The transcriptional start site of CiADAR1a is mapped within the truncated exon 2. CiADAR1a promoter is 1303 bp in length containing 4 IRF-Es. In the present study, we constructed pcDNA3.1 eukaryotic expression vectors with IRF1 and IRF3 and co-transfected them with pGL3-CiADAR1a promoter into CIK cells. The results showed that neither the over-expression of IRF1 or IRF3 nor Poly I:C stimulation significantly impacted CiADAR1a promoter activity in CIK cells. Together, according to the molecular and expression characteristics, subcellular localization and transcriptional

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

    Directory of Open Access Journals (Sweden)

    Katja Meyer

    2015-07-01

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

  18. Genetic Variation of Pre-mRNA Alternative Splicing in Human Populations

    OpenAIRE

    Lu, Zhi-xiang; Jiang, Peng; Xing, Yi

    2011-01-01

    The precise splicing outcome of a transcribed gene is controlled by complex interactions between cis regulatory splicing signals and trans-acting regulators. In higher eukaryotes, alternative splicing is a prevalent mechanism for generating transcriptome and proteome diversity. Alternative splicing can modulate gene function, affect organismal phenotype and cause disease. Common genetic variation that affects splicing regulation can lead to differences in alternative splicing between human in...

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

    OpenAIRE

    Long Ma; Xiaoyang Gao; Jintao Luo; Liange Huang; Yanling Teng; H Robert Horvitz

    2012-01-01

    RNA splicing is a major regulatory mechanism for controlling eukaryotic gene expression. By generating various splice isoforms from a single pre-mRNA, alternative splicing plays a key role in promoting the evolving complexity of metazoans. Numerous splicing factors have been identified. However, the in vivo functions of many splicing factors remain to be understood. In vivo studies are essential for understanding the molecular mechanisms of RNA splicing and the biology of numerous RNA splicin...

  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. 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. PMID:26992833

  2. Identification of four genes involved in suppression of the pre-mRNA splicing defect in the sng1-1/rhp6- mutant of fission yeast

    Indian Academy of Sciences (India)

    Alpana Naresh; Jagmohan Singh

    2000-01-01

    Apart from the global regulators of silencing in the fission yeast Schizosaccharomyces pombe, namely swi6, clr1, clr2, clr3, clr4 and rik1, the DNA repair gene rhp6 plays a unique role in mating-type silencing. Recently, we showed that sng1-1, a mutation in the 5′ splice junction of the second intron of the rhp6 gene, leads to derepression of both the silent loci mat2 and mat3 in switching background. To address the mechanism of rhp6 in silencing, we have isolated several extragenic suppressors of the sng1-1/rhp6- mutation. These suppressors fall into four complementation groups and are referred to as suppressor of rhp6: sur1, sur2, sur3 and sur4. Interestingly, reverse transcriptase polymerase chain reaction analysis of the rhp6 transcript shows that in contrast to about > 50% level of unspliced rhp6 pre-mRNA in the sng1-1/rhp6- mutant, there is a restoration of normal splicing to varying degrees in the suppressors. The sur2 gene belongs to the AAA-ATPase family of proteins, with maximum homology to the SIN1-associated protein SAP1 of Saccharomyces cerevisiae. We propose that sur2, along with sur1, sur3 and sur4, may play an as yet uncharacterized role in pre-mRNA splicing.

  3. A temperature-sensitive allele of a putative mRNA splicing helicase down-regulates many cell wall genes and causes radial swelling in Arabidopsis thaliana.

    Science.gov (United States)

    Howles, Paul A; Gebbie, Leigh K; Collings, David A; Varsani, Arvind; Broad, Ronan C; Ohms, Stephen; Birch, Rosemary J; Cork, Ann H; Arioli, Tony; Williamson, Richard E

    2016-05-01

    The putative RNA helicase encoded by the Arabidopsis gene At1g32490 is a homolog of the yeast splicing RNA helicases Prp2 and Prp22. We isolated a temperature-sensitive allele (rsw12) of the gene in a screen for root radial swelling mutants. Plants containing this allele grown at the restrictive temperature showed weak radial swelling, were stunted with reduced root elongation, and contained reduced levels of cellulose. The role of the protein was further explored by microarray analysis. By using both fold change cutoffs and a weighted gene coexpression network analysis (WGCNA) to investigate coexpression of genes, we found that the radial swelling phenotype was not linked to genes usually associated with primary cell wall biosynthesis. Instead, the mutation has strong effects on expression of secondary cell wall related genes. Many genes potentially associated with secondary walls were present in the most significant WGCNA module, as were genes coding for arabinogalactans and proteins with GPI anchors. The proportion of up-regulated genes that possess introns in rsw12 was above that expected if splicing was unrelated to the activity of the RNA helicase, suggesting that the helicase does indeed play a role in splicing in Arabidopsis. The phenotype may be due to a change in the expression of one or more genes coding for cell wall proteins. PMID:27008640

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

    Science.gov (United States)

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

    2016-01-01

    The Cystic Fibrosis p.Ile1234Val missense mutation actually creates a new dual splicing site possibly used either as a new acceptor or donor. Here, we aimed to test the accuracy of in silico predictions by comparing them with in vitro and ex vivo functional analyses of this mutation for an accurate CF diagnosis/prognosis. To this end, we applied a new in vitro strategy using a CFTR mini-gene which includes the complete CFTR coding sequence plus intron 22 (short version) which allows the assessment of alternatively spliced mRNA levels as well as the properties of the resulting abnormal CFTR protein regarding processing, intracellular localization and function. Our data demonstrate that p.Ile1234Val leads to usage of the alternative splicing donor (but not acceptor) resulting in alternative CFTR transcripts lacking 18 nts of exon 22 which produce a truncated CFTR protein with residual Cl- channel function. These results recapitulate data from native tissues of a CF patient. In conclusion, the existing in silico prediction models have limited application and ex vivo functional assessment of mutation effects should be made. Alternatively the in vitro strategy adopted here can be applied to assess the disease liability of mutations for an accurate CF diagnosis/prognosis. PMID:25735457

  5. The conserved splicing factor SUA controls alternative splicing of the developmental regulator ABI3 in Arabidopsis.

    NARCIS (Netherlands)

    Sugliani, M.; Brambilla, V.; Clerkx, E.J.M.; Koornneef, M.; Soppe, W.J.J.

    2010-01-01

    ABSCISIC ACID INSENSITIVE3 (ABI3) is a major regulator of seed maturation in Arabidopsis thaliana. We detected two ABI3 transcripts, ABI3- and ABI3-ß, which encode full-length and truncated proteins, respectively. Alternative splicing of ABI3 is developmentally regulated, and the ABI3-ß transcript a

  6. Individuals With Normal GLA Gene Sequence May Present Abnormally Spliced Alpha-Galactosidase mRNA Transcripts

    Directory of Open Access Journals (Sweden)

    Ferreira

    2015-12-01

    Full Text Available Background Deficient lysosomal α-galactosidase activity leads to intracellular accumulation of globotriaosylceramide (Gb3, which is the pathologic hallmark of Fabry disease (FD. There are over 750 pathogenic variants identified in the α-galactosidase gene (GLA. In rare patients, the cause of α-galactosidase deficiency is the overexpression of a GLA transcript with a cryptic exon in intron 4, which is physiologically present at trace levels. Objectives We aim to report abnormally spliced alpha-galactosidase mRNA transcripts found with a cDNA-based GLA genotyping protocol performed in 482 patients. Patients and Methods Genomic DNA and total RNA specimens were obtained from peripheral blood leukocytes of patients with premature stroke prospectively enrolled in the PORTYSTROKE study, or of patients with possible clinical manifestations of FD who have been referred for molecular diagnostic workup. Results Approximately 20% of the patients expressed alternatively spliced transcripts of GLA mRNA involving exon 3. We additionally report that such non-canonical transcripts are physiologically expressed at trace levels in healthy individuals, and that their expression in leukocytes markedly increased in blood samples kept at room-temperature for 48 hours before RNA extraction. Conclusions Production of alternatively spliced GLA transcripts might be involved in the regulation of GLA gene expression, and its deregulated overexpression, particularly if restricted to specific cells or tissues, might be the cause of organ-limited Gb3 pathology. Elucidation of the molecular mechanisms underlying the production of the non-canonical GLA transcripts warrants further investigation, as it may contribute important new data to the understanding of the molecular pathology of FD and Gb3-related disorders.

  7. A novel BRD4-NUT fusion in an undifferentiated sinonasal tumor highlights alternative splicing as a contributing oncogenic factor in NUT midline carcinoma.

    Science.gov (United States)

    Stirnweiss, A; McCarthy, K; Oommen, J; Crook, M L; Hardy, K; Kees, U R; Wilton, S D; Anazodo, A; Beesley, A H

    2015-01-01

    NUT midline carcinoma (NMC) is a fatal cancer that arises in various tissues along the upper midline of the body. The defining molecular feature of NMC is a chromosomal translocation that joins (in the majority of cases) the nuclear testis gene NUT (NUTM1) to the bromodomain protein family member 4 (BRD4) and thereby creating a fusion oncogene that disrupts cellular differentiation and drives the disease. In this study, we report the case of an adolescent NMC patient presenting with severe facial pain, proptosis and visual impairment due to a mass arising from the ethmoid sinus that invaded the right orbit and frontal lobe. Treatment involved radical resection, including exenteration of the affected eye with the view to consolidate treatment with radiation therapy; however, the patient experienced rapid tumor progression and passed away 79 days post resection. Molecular analysis of the tumor tissue identified a novel in-frame BRD4-NUT transcript, with BRD4 exon 15 fused to the last 124 nucleotides of NUT exon 2 (BRD4-NUT ex15:ex2Δnt1-585). The partial deletion of NUT exon 2 was attributed to a mid-exonic genomic breakpoint and the subsequent activation of a cryptic splice site further downstream within the exon. Inhibition of the canonical 3' acceptor splice site of NUT intron 1 in cell lines expressing the most common NMC fusion transcripts (PER-403, BRD4-NUT ex11:ex2; PER-624, BRD4-NUT ex15:ex2) induced alternative splicing from the same cryptic splice site as identified in the patient. Detection of low levels of an in-frame BRD4-NUT ex11:ex2Δnt1-585 transcript in PER-403 confirmed endogenous splicing from this alternative exon 2 splice site. Although further studies are necessary to assess the clinical relevance of the increasing number of variant fusions described in NMC, the findings presented in this case identify alternative splicing as a mechanism that contributes to this pathogenic complexity. PMID:26551281

  8. Splice connector with internal heat transfer jacket

    Science.gov (United States)

    Silva, Frank A.; Mayer, Robert W.

    1977-01-01

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

  9. Rituximab responsive immune thrombocytopenic purpura in an adult with underlying autoimmune lymphoproliferative syndrome due to a splice-site mutation (IVS7+2 T>C) affecting the Fas gene

    OpenAIRE

    Wei, Andrew; Cowie, Tiffany

    2007-01-01

    A 36 yr-old man of Israeli descent with a history of childhood splenectomy for severe thrombocytopenia and a family history of autoimmune lymphoproliferative syndrome (ALPS), presented with severe immune thrombocytopenic purpura refractory to standard therapy. He was found to possess a heterozygous mutation in the Fas gene (also termed TNFRSF6, CD95, Apo-1) affecting the donor splice site of intron 7 (IVS7+2 T>C). This frameshift mutation truncates the cytoplasmic domain of the Fas death rece...

  10. Exon Expression and Alternatively Spliced Genes in Tourette Syndrome

    NARCIS (Netherlands)

    Tian, Yingfang; Liao, Isaac H.; Zhan, Xinhua; Gunther, Joan R.; Ander, Bradley P.; Liu, Dazhi; Lit, Lisa; Jickling, Glen C.; Corbett, Blythe A.; Bos-Veneman, Netty G. P.; Hoekstra, Pieter J.; Sharp, Frank R.

    2011-01-01

    Tourette Syndrome (TS) is diagnosed based upon clinical criteria including motor and vocal tics. We hypothesized that differences in exon expression and splicing might be useful for pathophysiology and diagnosis. To demonstrate exon expression and alternatively spliced gene differences in blood of i

  11. 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 alterna...

  12. Synaptic signaling and aberrant RNA splicing in autism spectrum disorders

    OpenAIRE

    Ryan M Smith; Wolfgang eSadee

    2011-01-01

    Interactions between presynaptic and postsynaptic cellular adhesion molecules drive synapse maturation during development. These trans-synaptic interactions are regulated by alternative splicing of cellular adhesion molecule RNAs, which ultimately determines neurotransmitter phenotype. The diverse assortment of RNAs produced by alternative splicing generates countless protein isoforms necessary for guiding specialized cell-to-cell connectivity. Failure to generate the appropriate synaptic ...

  13. Synaptic Signaling and Aberrant RNA Splicing in Autism Spectrum Disorders

    OpenAIRE

    Smith, Ryan M; Sadee, Wolfgang

    2011-01-01

    Interactions between presynaptic and postsynaptic cellular adhesion molecules (CAMs) drive synapse maturation during development. These trans-synaptic interactions are regulated by alternative splicing of CAM RNAs, which ultimately determines neurotransmitter phenotype. The diverse assortment of RNAs produced by alternative splicing generates countless protein isoforms necessary for guiding specialized cell-to-cell connectivity. Failure to generate the appropriate synaptic adhesion proteins i...

  14. Quantitative regulation of alternative splicing in evolution and development

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob L; Roy, Scott W;

    2009-01-01

    Alternative splicing (AS) is a widespread mechanism with an important role in increasing transcriptome and proteome diversity by generating multiple different products from the same gene. Evolutionary studies of AS have focused primarily on the conservation of alternatively spliced sequences or of...... additional layer in complex gene regulatory networks and in the emergence of genetic novelties....

  15. Tissue-specific splicing factor gene expression signatures

    NARCIS (Netherlands)

    A.R. Grosso; A.Q. Gomes (Anita); N.L. Barbosa-Morais (Nuno); S. Caldeira (Sandra); N.P. Thorne (Natalie); G. Grech (Godfrey); M.M. von Lindern (Marieke); M. Carmo-Fonseca (Maria)

    2008-01-01

    textabstractThe alternative splicing code that controls and coordinates the transcriptome in complex multicellular organisms remains poorly understood. It has long been argued that regulation of alternative splicing relies on combinatorial interactions between multiple proteins, and that tissue-spec

  16. Recent mobility of plastid encoded group II introns and twintrons in five strains of the unicellular red alga Porphyridium

    Directory of Open Access Journals (Sweden)

    Marie-Mathilde Perrineau

    2015-06-01

    Full Text Available Group II introns are closely linked to eukaryote evolution because nuclear spliceosomal introns and the small RNAs associated with the spliceosome are thought to trace their ancient origins to these mobile elements. Therefore, elucidating how group II introns move, and how they lose mobility can potentially shed light on fundamental aspects of eukaryote biology. To this end, we studied five strains of the unicellular red alga Porphyridium purpureum that surprisingly contain 42 group II introns in their plastid genomes. We focused on a subset of these introns that encode mobility-conferring intron-encoded proteins (IEPs and found them to be distributed among the strains in a lineage-specific manner. The reverse transcriptase and maturase domains were present in all lineages but the DNA endonuclease domain was deleted in vertically inherited introns, demonstrating a key step in the loss of mobility. P. purpureum plastid intron RNAs had a classic group IIB secondary structure despite variability in the DIII and DVI domains. We report for the first time the presence of twintrons (introns-within-introns, derived from the same mobile element in Rhodophyta. The P. purpureum IEPs and their mobile introns provide a valuable model for the study of mobile retroelements in eukaryotes and offer promise for biotechnological applications.

  17. Splice mutations preserve myophosphorylase activity that ameliorates the phenotype in McArdle disease

    DEFF Research Database (Denmark)

    Vissing, John; Duno, Morten; Schwartz, Marianne;

    2009-01-01

    features of two patients with a variant form of McArdle disease, associated with unusually high exercise capacity. Physiologic findings were compared to those in 47 patients with typical McArdle disease, and 17 healthy subjects. Subjects performed an ischaemic forearm exercise test to assess lactate...... and ammonia production. Peak oxidative capacity (VO2max) and cardiac output were determined, using cycle ergometry as the exercise modality. The two patients with atypical McArdle disease carried common mutations on one allele (R50X and G205S), and novel splice mutations in introns 3 [IVS3-26A>G (c.425-26A......>G)] and 5 [IVS5-601G>A (c.856-601G>A)] on the other allele. Plasma lactate after ischaemic exercise decreased in all typical McArdle patients, but increased in the two atypical McArdle patients (10% of that in healthy subjects). Peak workload and oxidative capacity were 2-fold higher in patients...

  18. 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...

  19. 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.

  20. 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

    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...... 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...