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Sample records for human spliceosomal u1

  1. Therapeutic activity of modified U1 core spliceosomal particles

    PubMed Central

    Rogalska, Malgorzata Ewa; Tajnik, Mojca; Licastro, Danilo; Bussani, Erica; Camparini, Luca; Mattioli, Chiara; Pagani, Franco

    2016-01-01

    Modified U1 snRNAs bound to intronic sequences downstream of the 5′ splice site correct exon skipping caused by different types of mutations. Here we evaluate the therapeutic activity and structural requirements of these exon-specific U1 snRNA (ExSpeU1) particles. In a severe spinal muscular atrophy, mouse model, ExSpeU1, introduced by germline transgenesis, increases SMN2 exon 7 inclusion, SMN protein production and extends life span. In vitro, RNA mutant analysis and silencing experiments show that while U1A protein is dispensable, the 70K and stem loop IV elements mediate most of the splicing rescue activity through improvement of exon and intron definition. Our findings indicate that precise engineering of the U1 core spliceosomal RNA particle has therapeutic potential in pathologies associated with exon-skipping mutations. PMID:27041075

  2. Therapeutic activity of modified U1 core spliceosomal particles.

    PubMed

    Rogalska, Malgorzata Ewa; Tajnik, Mojca; Licastro, Danilo; Bussani, Erica; Camparini, Luca; Mattioli, Chiara; Pagani, Franco

    2016-01-01

    Modified U1 snRNAs bound to intronic sequences downstream of the 5' splice site correct exon skipping caused by different types of mutations. Here we evaluate the therapeutic activity and structural requirements of these exon-specific U1 snRNA (ExSpeU1) particles. In a severe spinal muscular atrophy, mouse model, ExSpeU1, introduced by germline transgenesis, increases SMN2 exon 7 inclusion, SMN protein production and extends life span. In vitro, RNA mutant analysis and silencing experiments show that while U1A protein is dispensable, the 70K and stem loop IV elements mediate most of the splicing rescue activity through improvement of exon and intron definition. Our findings indicate that precise engineering of the U1 core spliceosomal RNA particle has therapeutic potential in pathologies associated with exon-skipping mutations. PMID:27041075

  3. Molecular characterization of the spliceosomal proteins U1A and U2B" from higher plants.

    PubMed Central

    Simpson, G G; Clark, G P; Rothnie, H M; Boelens, W; van Venrooij, W; Brown, J W

    1995-01-01

    In addition to their role in pre-mRNA splicing, the human spliceosomal proteins U1A and U2B" are important models of how RNP motif-containing proteins execute sequence-specific RNA binding. Genes encoding U1A and U2B" have been isolated from potato and thereby provide the only evolutionary comparison available for both proteins and represent the only full-length genes encoding plant spliceosomal proteins to have been cloned and characterized. In vitro RNA binding experiments revealed the ability of potato U2B" to interact with human U2A' to enhance sequence-specific binding and to distinguish cognate RNAs of either plant or animal origin. A comparison of the sequence of U1A and U2B" proteins indicated that multiple residues which could affect RNP motif conformation probably govern the specific distinction in RNA binding by these proteins. Since human U1A modulates polyadenylation in vertebrates, the possibility that plant U1A might be exploited in the characterization of this process in plants was examined. However, unlike vertebrate U1A, neither U1A from potato nor Arabidopsis bound their own mRNA and no evidence for binding to upstream efficiency elements in polyadenylation signals was obtained, suggesting that plant U1A is not involved in polyadenylation. Images PMID:7556097

  4. Structural bioinformatics of the human spliceosomal proteome

    PubMed Central

    Korneta, Iga; Magnus, Marcin; Bujnicki, Janusz M.

    2012-01-01

    In this work, we describe the results of a comprehensive structural bioinformatics analysis of the spliceosomal proteome. We used fold recognition analysis to complement prior data on the ordered domains of 252 human splicing proteins. Examples of newly identified domains include a PWI domain in the U5 snRNP protein 200K (hBrr2, residues 258–338), while examples of previously known domains with a newly determined fold include the DUF1115 domain of the U4/U6 di-snRNP protein 90K (hPrp3, residues 540–683). We also established a non-redundant set of experimental models of spliceosomal proteins, as well as constructed in silico models for regions without an experimental structure. The combined set of structural models is available for download. Altogether, over 90% of the ordered regions of the spliceosomal proteome can be represented structurally with a high degree of confidence. We analyzed the reduced spliceosomal proteome of the intron-poor organism Giardia lamblia, and as a result, we proposed a candidate set of ordered structural regions necessary for a functional spliceosome. The results of this work will aid experimental and structural analyses of the spliceosomal proteins and complexes, and can serve as a starting point for multiscale modeling of the structure of the entire spliceosome. PMID:22573172

  5. Comprehensive proteomic analysis of the human spliceosome

    NASA Astrophysics Data System (ADS)

    Zhou, Zhaolan; Licklider, Lawrence J.; Gygi, Steven P.; Reed, Robin

    2002-09-01

    The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins and salt-stable spliceosome `core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify ~145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.

  6. Large-Scale Proteomic Analysis of the Human Spliceosome

    PubMed Central

    Rappsilber, Juri; Ryder, Ursula; Lamond, Angus I.; Mann, Matthias

    2002-01-01

    In a previous proteomic study of the human spliceosome, we identified 42 spliceosome-associated factors, including 19 novel ones. Using enhanced mass spectrometric tools and improved databases, we now report identification of 311 proteins that copurify with splicing complexes assembled on two separate pre-mRNAs. All known essential human splicing factors were found, and 96 novel proteins were identified, of which 55 contain domains directly linking them to functions in splicing/RNA processing. We also detected 20 proteins related to transcription, which indicates a direct connection between this process and splicing. This investigation provides the most detailed inventory of human spliceosome-associated factors to date, and the data indicate a number of interesting links coordinating splicing with other steps in the gene expression pathway. PMID:12176931

  7. Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

    PubMed Central

    Möhlmann, Sina; Mathew, Rebecca; Neumann, Piotr; Schmitt, Andreas; Lührmann, Reinhard; Ficner, Ralf

    2014-01-01

    The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP. PMID:24914973

  8. Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

    SciTech Connect

    Möhlmann, Sina; Mathew, Rebecca; Neumann, Piotr; Schmitt, Andreas; Lührmann, Reinhard; Ficner, Ralf

    2014-06-01

    The crystal structure of the helicase domain of the human spliceosomal DEAD-box protein Prp28 was solved by SAD. The binding of ADP and ATP by Prp28 was studied biochemically and analysed with regard to the crystal structure. The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

  9. Crystallization and biochemical characterization of the human spliceosomal Aar2-Prp8(RNaseH) complex.

    PubMed

    Santos, Karine; Preussner, Marco; Heroven, Anna Christina; Weber, Gert

    2015-11-01

    In eukaryotes, the removal of nuclear noncoding sequences (pre-mRNA splicing) is catalyzed by the spliceosome, which consists of five ribonucleoprotein particles (U1, U2, U4, U5 and U6 snRNPs, each with a respective snRNA) and a plethora of protein factors that aid spliceosomal maturation, assembly, activation and disassembly. Recently, the U5 snRNP maturation factor Aar2p from baker's yeast has been characterized structurally and biochemically. Aar2p binds to the RNaseH (RH) and Jab1/MPN domains of the highly conserved U5-specific Prp8p, which forms a framework for the spliceosomal catalytic centre. Thereby, Aar2p sterically excludes Brr2p, a helicase essential for the catalytic activation of the spliceosome, from Prp8p binding. At the same time, Aar2p blocks U4/U6 di-snRNA binding to Prp8p. Aar2p therefore prevents premature spliceosome activation and its functions are regulated by reversible phosphorylation. To date, little is known about the hypothetical human Aar2 (hsAar2) orthologue C20ORF4. This study identifies C20ORF4 (i) as part of the HeLa proteome by Western blotting and (ii) as a true Aar2 orthologue which binds to the RH domain (hsRH) of Prp8 and corroborates an evolutionary link between yeast and human Aar2 function. An elaborate strategy was devised to crystallize hsAar2 in complex with hsRH. The analysis of initial weakly diffracting crystals obtained by in situ proteolysis and homology modelling guided the design of an hsAar2 construct in which an internal loop was replaced by three serines (hsAar2(Δloop)). A complex of hsAar2(Δloop) and hsRH crystallized in space group C2; the crystals diffracted to 2.35 Å resolution and were suitable for structure determination by molecular-replacement approaches. The study presented here suggests a connection between Aar2 and the spliceosome in human cells and paves the way for structural studies of human Aar2. PMID:26527271

  10. Functional mammalian spliceosomal complex E contains SMN complex proteins in addition to U1 and U2 snRNPs

    PubMed Central

    Makarov, Evgeny M.; Owen, Nicholas; Bottrill, Andrew; Makarova, Olga V.

    2012-01-01

    Spliceosomes remove introns from primary gene transcripts. They assemble de novo on each intron through a series of steps that involve the incorporation of five snRNP particles and multiple non-snRNP proteins. In mammals, all the intermediate complexes have been characterized on one transcript (MINX), with the exception of the very first, complex E. We have purified this complex by two independent procedures using antibodies to either U1-A or PRPF40A proteins, which are known to associate at an early stage of assembly. We demonstrate that the purified complexes are functional in splicing using commitment assays. These complexes contain components expected to be in the E complex and a number of previously unrecognized factors, including survival of motor neurons (SMN) and proteins of the SMN-associated complex. Depletion of the SMN complex proteins from nuclear extracts inhibits formation of the E complex and causes non-productive complexes to accumulate. This suggests that the SMN complex stabilizes the association of U1 and U2 snRNPs with pre-mRNA. In addition, the antibody to PRPF40A precipitated U2 snRNPs from nuclear extracts, indicating that PRPF40A associates with U2 snRNPs. PMID:22110043

  11. Major spliceosome defects cause male infertility and are associated with nonobstructive azoospermia in humans.

    PubMed

    Wu, Hao; Sun, Liwei; Wen, Yang; Liu, Yujuan; Yu, Jun; Mao, Feiyu; Wang, Ya; Tong, Chao; Guo, Xuejiang; Hu, Zhibin; Sha, Jiahao; Liu, Mingxi; Xia, Laixin

    2016-04-12

    Processing of pre-mRNA into mRNA is an important regulatory mechanism in eukaryotes that is mediated by the spliceosome, a huge and dynamic ribonucleoprotein complex. Splicing defects are implicated in a spectrum of human disease, but the underlying mechanistic links remain largely unresolved. Using a genome-wide association approach, we have recently identified single nucleotide polymorphisms in humans that associate with nonobstructive azoospermia (NOA), a common cause of male infertility. Here, using genetic manipulation of corresponding candidate loci in Drosophila, we show that the spliceosome component SNRPA1/U2A is essential for male fertility. Loss of U2A in germ cells of the Drosophila testis does not affect germline stem cells, but does result in the accumulation of mitotic spermatogonia that fail to differentiate into spermatocytes and mature sperm. Lack of U2A causes insufficient splicing of mRNAs required for the transition of germ cells from proliferation to differentiation. We show that germ cell-specific disruption of other components of the major spliceosome manifests with the same phenotype, demonstrating that mRNA processing is required for the differentiation of spermatogonia. This requirement is conserved, and expression of human SNRPA1 fully restores spermatogenesis in U2A mutant flies. We further report that several missense mutations in human SNRPA1 that inhibit the assembly of the major spliceosome dominantly disrupt spermatogonial differentiation in Drosophila. Collectively, our findings uncover a conserved and specific requirement for the major spliceosome during the transition from spermatogonial proliferation to differentiation in the male testis, suggesting that spliceosome defects affecting the differentiation of human spermatogonia contribute to NOA. PMID:27035939

  12. Spliceosomal Immunophilins

    PubMed Central

    Mesa, Annia; Somarelli, Jason A.; Herrera, Rene J.

    2008-01-01

    The spliceosome is a dynamic, macromolecular complex, which removes non-protein-coding introns from pre-mRNA to form mature mRNA in a process known as splicing. This ribonucleoprotein assembly is comprised of five uridine-rich small nuclear RNAs (snRNAs) as well as over 300 proteins. In humans, several of the known splicing factors are members of the immunophilin superfamily. Immunophilins are peptidyl-prolyl cis-trans isomerases that catalyze the conversion of proteins from cis to trans at Xaa-Pro bonds. Our review of the data portrays a picture of this protein family as activators of spliceosomal proteins by way of folding and transport. PMID:18544344

  13. Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16.

    PubMed

    Martínez-Salazar, Martha; López-Urrutia, Eduardo; Arechaga-Ocampo, Elena; Bonilla-Moreno, Raul; Martínez-Castillo, Macario; Díaz-Hernández, Job; Del Moral-Hernández, Oscar; Cedillo-Barrón, Leticia; Martines-Juarez, Víctor; De Nova-Ocampo, Monica; Valdes, Jesús; Berumen, Jaime; Villegas-Sepúlveda, Nicolás

    2014-12-01

    The human papillomavirus type 16 (HPV-16) E6/E7 spliced transcripts are heterogeneously expressed in cervical carcinoma. The heterogeneity of the E6/E7 splicing profile might be in part due to the intrinsic variation of splicing factors in tumor cells. However, the splicing factors that bind the E6/E7 intron 1 (In-1) have not been defined. Therefore, we aimed to identify these factors; we used HeLa nuclear extracts (NE) for in vitro spliceosome assembly. The proteins were allowed to bind to an RNA/DNA hybrid formed by the In-1 transcript and a 5'-biotinylated DNA oligonucleotide complementary to the upstream exon sequence, which prevented interference in protein binding to the intron. The hybrid probes bound with the nuclear proteins were coupled to streptavidin magnetic beads for chromatography affinity purification. Proteins were eluted and identified by mass spectrometry (MS). Approximately 170 proteins were identified by MS, 80% of which were RNA binding proteins, including canonical spliceosome core components, helicases and regulatory splicing factors. The canonical factors were identified as components of the spliceosomal B-complex. Although 35-40 of the identified factors were cognate splicing factors or helicases, they have not been previously detected in spliceosome complexes that were assembled using in vivo or in vitro models. PMID:25108200

  14. Cwc2 and its human homologue RBM22 promote an active conformation of the spliceosome catalytic centre

    PubMed Central

    Rasche, Nicolas; Dybkov, Olexandr; Schmitzová, Jana; Akyildiz, Berktan; Fabrizio, Patrizia; Lührmann, Reinhard

    2012-01-01

    RNA-structural elements play key roles in pre-mRNA splicing catalysis; yet, the formation of catalytically competent RNA structures requires the assistance of spliceosomal proteins. We show that the S. cerevisiae Cwc2 protein functions prior to step 1 of splicing, and it is not required for the Prp2-mediated spliceosome remodelling that generates the catalytically active B* complex, suggesting that Cwc2 plays a more sophisticated role in the generation of a functional catalytic centre. In active spliceosomes, Cwc2 contacts catalytically important RNA elements, including the U6 internal stem-loop (ISL), and regions of U6 and the pre-mRNA intron near the 5′ splice site, placing Cwc2 at/near the spliceosome's catalytic centre. These interactions are evolutionarily conserved, as shown by studies with Cwc2's human counterpart RBM22, indicating that Cwc2/RBM22–RNA contacts are functionally important. We propose that Cwc2 induces an active conformation of the spliceosome's catalytic RNA elements. Thus, the function of RNA–RNA tertiary interactions within group II introns, namely to induce an active conformation of domain V, may be fulfilled by proteins that contact the functionally analogous U6-ISL, within the spliceosome. PMID:22246180

  15. Functional organization of the Sm core in the crystal structure of human U1 snRNP

    PubMed Central

    Weber, Gert; Trowitzsch, Simon; Kastner, Berthold; Lührmann, Reinhard; Wahl, Markus C

    2010-01-01

    U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5′-splice site early during spliceosome assembly. It represents a prototype spliceosomal subunit containing a paradigmatic Sm core RNP. The crystal structure of human U1 snRNP obtained from natively purified material by in situ limited proteolysis at 4.4 Å resolution reveals how the seven Sm proteins, each recognize one nucleotide of the Sm site RNA using their Sm1 and Sm2 motifs. Proteins D1 and D2 guide the snRNA into and out of the Sm ring, and proteins F and E mediate a direct interaction between the Sm site termini. Terminal extensions of proteins D1, D2 and B/B′, and extended internal loops in D2 and B/B′ support a four-way RNA junction and a 3′-terminal stem-loop on opposite sides of the Sm core RNP, respectively. On a higher organizational level, the core RNP presents multiple attachment sites for the U1-specific 70K protein. The intricate, multi-layered interplay of proteins and RNA rationalizes the hierarchical assembly of U snRNPs in vitro and in vivo. PMID:21113136

  16. Spliceosomal intronogenesis.

    PubMed

    Lee, Sujin; Stevens, Scott W

    2016-06-01

    The presence of intervening sequences, termed introns, is a defining characteristic of eukaryotic nuclear genomes. Once transcribed into pre-mRNA, these introns must be removed within the spliceosome before export of the processed mRNA to the cytoplasm, where it is translated into protein. Although intron loss has been demonstrated experimentally, several mysteries remain regarding the origin and propagation of introns. Indeed, documented evidence of gain of an intron has only been suggested by phylogenetic analyses. We report the use of a strategy that detects selected intron gain and loss events. We have experimentally verified, to our knowledge, the first demonstrations of intron transposition in any organism. From our screen, we detected two separate intron gain events characterized by the perfect transposition of a reporter intron into the yeast genes RPL8B and ADH2, respectively. We show that the newly acquired introns are able to be removed from their respective pre-mRNAs by the spliceosome. Additionally, the novel allele, RPL8Bint, is functional when overexpressed within the genome in a strain lacking the Rpl8 paralogue RPL8A, demonstrating that the gene targeted for intronogenesis is functional. PMID:27217561

  17. A human RNA helicase-like protein, HRH1, facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome.

    PubMed

    Ohno, M; Shimura, Y

    1996-04-15

    Because the nuclear export of mRNA occurs only after the splicing reaction is completed, intron-containing pre-mRNA does not normally appear in the cytoplasm. As a mechanism to secure this, intron-containing RNA is retained in the nucleus via formation of the spliceosome. Therefore, the process of releasing spliced mRNA from the spliceosome after completion of splicing is an essential step for triggering the nuclear export of the spliced mRNA. In budding yeast, RNA helicase-like protein Prp22 is implicated in this process. Here we demonstrate the function of HRH1, a human protein homologous to Prp22, in mammalian cells using dominant-negative HRH1++ mutants (dn-HRH1). dn-HRH1 protein stalls on the spliceosome and prevents release of the spliced RNA from the spliceosome in vitro. Expression of dn-HRH1 in mammalian cells leads to inhibition of splicing and to extensive nuclear export of unspliced pre-mRNA, probably because of the incapability of recycling spliceosome components that normally retain the pre-mRNA in the nucleus. The arginine/serine-rich domain (RS domain) of HRH1, which is missing in Prp22, confers a nuclear localization signal, and appears to facilitate the interaction of HRH1 with the spliceosome. This is the first report on a bona fide mammalian homolog of yeast Prp splicing factor, and also on a mammalian RNA helicase-like splicing factor. PMID:8608946

  18. Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome.

    PubMed

    Schütze, Tonio; Ulrich, Alexander K C; Apelt, Luise; Will, Cindy L; Bartlick, Natascha; Seeger, Martin; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C

    2016-02-01

    Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein-protein interaction platform that might organize the relative positioning of other proteins during splicing. PMID:26673105

  19. The N-terminus of Prp1 (Prp6/U5-102 K) is essential for spliceosome activation in vivo

    PubMed Central

    Lützelberger, Martin; Bottner, Claudia A.; Schwelnus, Wiebke; Zock-Emmenthal, Susanne; Razanau, Aleh; Käufer, Norbert F.

    2010-01-01

    The spliceosomal protein Prp1 (Prp6/U5-102 K) is necessary for the integrity of pre-catalytic spliceosomal complexes. We have identified a novel regulatory function for Prp1. Expression of mutations in the N-terminus of Prp1 leads to the accumulation of pre-catalytic spliceosomal complexes containing the five snRNAs U1, U2, U5 and U4/U6 and pre-mRNAs. The mutations in the N-terminus, which prevent splicing to occur, include in vitro and in vivo identified phosphorylation sites of Prp4 kinase. These sites are highly conserved in the human ortholog U5-102 K. The results presented here demonstrate that structural integrity of the N-terminus is required to mediate a splicing event, but is not necessary for the assembly of spliceosomes. PMID:20007600

  20. Primary structure of a human arginine-rich nuclear protein that colocalizes with spliceosome components

    SciTech Connect

    Chaudhary, N.; McMahon, C.; Blobel, G. )

    1991-09-15

    The cDNA for a 54-kDa nuclear protein (p54) has been cloned from a human hepatoma expression library. Contained within p54 is an arginine/serine-rich region similar to segments of several proteins that participate in pre-mRNA splicing including the 70-kDa component of U1 small nuclear ribonucleoprotein particle (snRNP) and the Drosophila transformer and suppressor-of-white-apricot proteins. The arginine/serine-rich region is dominated by a series of 8-amino acid imperfect repetitive motifs (consensus sequence, Arg-Arg-Ser-Arg-Ser-Arg-Ser-Arg). Antibodies raised against synthetic peptides of p54 react with an {approximately}70-kDa protein on immunoblots of HeLa cell and rat liver nuclear proteins. This apparent discrepancy in mass is also observed when p54 mRNA is translated in vitro. Indirect immunofluorescence studies in HeLa cells show that p54 is distributed throughout the nucleus in a speckled pattern, with an additional diffuse labeling of the nucleus excluding the nucleoli. Double immunofluorescence experiments indicate that these punctate regions are coincident with the speckles seen in cells stained with antibodies against several constituents of the pre-mRNA splicing machinery. Sedimentation analysis of HeLa cell extracts on sucrose gradients showed that p54 migrates at 4-6 S, indicating that the protein is not a tightly associated component of snRNPs. Although the function of p54 is not yet known, the structure and immunolocalization data suggest that this protein may have a role in pre-mRNA processing.

  1. CryoEM structures of two spliceosomal complexes: starter and dessert at the spliceosome feast

    PubMed Central

    Nguyen, Thi Hoang Duong; Galej, Wojciech P; Fica, Sebastian M; Lin, Pei-Chun; Newman, Andrew J; Nagai, Kiyoshi

    2016-01-01

    The spliceosome is formed on pre-mRNA substrates from five small nuclear ribonucleoprotein particles (U1, U2, U4/U6 and U5 snRNPs), and numerous non-snRNP factors. Saccharomyces cerevisiae U4/U6.U5 tri-snRNP comprises U5 snRNA, U4/U6 snRNA duplex and approximately 30 proteins and represents a substantial part of the spliceosome before activation. Schizosaccharomyces pombe U2.U6.U5 spliceosomal complex is a post-catalytic intron lariat spliceosome containing U2 and U5 snRNPs, NTC (nineteen complex), NTC-related proteins (NTR), U6 snRNA, and an RNA intron lariat. Two recent papers describe near-complete atomic structures of these complexes based on cryoEM single-particle analysis. The U4/U6.U5 tri-snRNP structure provides crucial insight into the activation mechanism of the spliceosome. The U2.U6.U5 complex reveals the striking architecture of NTC and NTR and important features of the group II intron-like catalytic RNA core remaining after spliced mRNA is released. These two structures greatly advance our understanding of the mechanism of pre-mRNA splicing. PMID:26803803

  2. CryoEM structures of two spliceosomal complexes: starter and dessert at the spliceosome feast.

    PubMed

    Nguyen, Thi Hoang Duong; Galej, Wojciech P; Fica, Sebastian M; Lin, Pei-Chun; Newman, Andrew J; Nagai, Kiyoshi

    2016-02-01

    The spliceosome is formed on pre-mRNA substrates from five small nuclear ribonucleoprotein particles (U1, U2, U4/U6 and U5 snRNPs), and numerous non-snRNP factors. Saccharomyces cerevisiae U4/U6.U5 tri-snRNP comprises U5 snRNA, U4/U6 snRNA duplex and approximately 30 proteins and represents a substantial part of the spliceosome before activation. Schizosaccharomyces pombe U2.U6.U5 spliceosomal complex is a post-catalytic intron lariat spliceosome containing U2 and U5 snRNPs, NTC (nineteen complex), NTC-related proteins (NTR), U6 snRNA, and an RNA intron lariat. Two recent papers describe near-complete atomic structures of these complexes based on cryoEM single-particle analysis. The U4/U6.U5 tri-snRNP structure provides crucial insight into the activation mechanism of the spliceosome. The U2.U6.U5 complex reveals the striking architecture of NTC and NTR and important features of the group II intron-like catalytic RNA core remaining after spliced mRNA is released. These two structures greatly advance our understanding of the mechanism of pre-mRNA splicing. PMID:26803803

  3. Splicing diversity revealed by reduced spliceosomes in C. merolae and other organisms

    PubMed Central

    Hudson, Andrew J; Stark, Martha R; Fast, Naomi M; Russell, Anthony G; Rader, Stephen D

    2015-01-01

    Pre-mRNA splicing has been considered one of the hallmarks of eukaryotes, yet its diversity is astonishing: the number of substrate introns for splicing ranges from hundreds of thousands in humans to a mere handful in certain parasites. The catalytic machinery that carries out splicing, the spliceosome, is similarly diverse, with over 300 associated proteins in humans to a few tens in other organisms. In this Point of View, we discuss recent work characterizing the reduced spliceosome of the acidophilic red alga Cyanidioschyzon merolae, which further highlights the diversity of splicing in that it does not possess the U1 snRNP that is characteristically responsible for 5′ splice site recognition. Comparisons to other organisms with reduced spliceosomes, such as microsporidia, trypanosomes, and Giardia, help to identify the most highly conserved splicing factors, pointing to the essential core of this complex machine. These observations argue for increased exploration of important biochemical processes through study of a wider ranger of organisms. PMID:26400738

  4. The Arabidopsis homolog of human minor spliceosomal protein U11-48K plays a crucial role in U12 intron splicing and plant development

    PubMed Central

    Xu, Tao; Kim, Bo Mi; Kwak, Kyung Jin; Jung, Hyun Ju; Kang, Hunseung

    2016-01-01

    The minor U12 introns are removed from precursor mRNAs by the U12 intron-specific minor spliceosome. Among the seven ribonucleoproteins unique to the minor spliceosome, denoted as U11/U12-20K, U11/U12-25K, U11/U12-31K, U11/U12-65K, U11-35K, U11-48K, and U11-59K, the roles of only U11/U12-31K and U11/U12-65K have been demonstrated in U12 intron splicing and plant development. Here, the functional role of the Arabidopsis homolog of human U11-48K in U12 intron splicing and the development of Arabidopsis thaliana was examined using transgenic knockdown plants. The u11-48k mutants exhibited several defects in growth and development, such as severely arrested primary inflorescence stems, formation of serrated leaves, production of many rosette leaves after bolting, and delayed senescence. The splicing of most U12 introns analyzed was impaired in the u11-48k mutants. Comparative analysis of the splicing defects and phenotypes among the u11/u12-31k, u11-48k, and u11/12-65k mutants showed that the severity of abnormal development was closely correlated with the degree of impairment in U12 intron splicing. Taken together, these results provide compelling evidence that the Arabidopsis homolog of human U11-48K protein, as well as U11/U12-31K and U11/U12-65K proteins, is necessary for correct splicing of U12 introns and normal plant growth and development. PMID:27091878

  5. The Arabidopsis homolog of human minor spliceosomal protein U11-48K plays a crucial role in U12 intron splicing and plant development.

    PubMed

    Xu, Tao; Kim, Bo Mi; Kwak, Kyung Jin; Jung, Hyun Ju; Kang, Hunseung

    2016-05-01

    The minor U12 introns are removed from precursor mRNAs by the U12 intron-specific minor spliceosome. Among the seven ribonucleoproteins unique to the minor spliceosome, denoted as U11/U12-20K, U11/U12-25K, U11/U12-31K, U11/U12-65K, U11-35K, U11-48K, and U11-59K, the roles of only U11/U12-31K and U11/U12-65K have been demonstrated in U12 intron splicing and plant development. Here, the functional role of the Arabidopsis homolog of human U11-48K in U12 intron splicing and the development of Arabidopsis thaliana was examined using transgenic knockdown plants. The u11-48k mutants exhibited several defects in growth and development, such as severely arrested primary inflorescence stems, formation of serrated leaves, production of many rosette leaves after bolting, and delayed senescence. The splicing of most U12 introns analyzed was impaired in the u11-48k mutants. Comparative analysis of the splicing defects and phenotypes among the u11/u12-31k, u11-48k, and u11/12-65k mutants showed that the severity of abnormal development was closely correlated with the degree of impairment in U12 intron splicing. Taken together, these results provide compelling evidence that the Arabidopsis homolog of human U11-48K protein, as well as U11/U12-31K and U11/U12-65K proteins, is necessary for correct splicing of U12 introns and normal plant growth and development. PMID:27091878

  6. RNAtomy of the Spliceosome's heart

    PubMed Central

    Bonnal, Sophie; Valcárcel, Juan

    2013-01-01

    The EMBO J 32: 2804–2818 10.1038/emboj.2013.198; published online 09032013 In his 1543 monumental work De humanis corpori fabrica, Andreas Vesalius used rigorous dissection practices and a mechanistic view of the organ's function to provide the first accurate anatomical description of a human heart. Guided by similar principles of meticulous structural probing and mechanistic explanatory potential, Anokhina and colleagues in this issue of The EMBO Journal dissect the molecular topology of the RNA heart of the Spliceosome, the ribonucleoprotein machinery in charge of intron removal. Their findings reveal key structural features with important implications for understanding the mechanisms of pre-mRNA splicing catalysis. PMID:24065126

  7. U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.

    PubMed

    Bai, Bing; Hales, Chadwick M; Chen, Ping-Chung; Gozal, Yair; Dammer, Eric B; Fritz, Jason J; Wang, Xusheng; Xia, Qiangwei; Duong, Duc M; Street, Craig; Cantero, Gloria; Cheng, Dongmei; Jones, Drew R; Wu, Zhiping; Li, Yuxin; Diner, Ian; Heilman, Craig J; Rees, Howard D; Wu, Hao; Lin, Li; Szulwach, Keith E; Gearing, Marla; Mufson, Elliott J; Bennett, David A; Montine, Thomas J; Seyfried, Nicholas T; Wingo, Thomas S; Sun, Yi E; Jin, Peng; Hanfelt, John; Willcock, Donna M; Levey, Allan; Lah, James J; Peng, Junmin

    2013-10-01

    Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of β-amyloid and tau in Alzheimer's disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis. PMID:24023061

  8. Crystal structure of a core spliceosomal protein interface.

    PubMed

    Schellenberg, Matthew J; Edwards, Ross A; Ritchie, Dustin B; Kent, Oliver A; Golas, Monika M; Stark, Holger; Lührmann, Reinhard; Glover, J N Mark; MacMillan, Andrew M

    2006-01-31

    The precise excision of introns from precursor mRNAs (pre-mRNAs) in eukaryotes is accomplished by the spliceosome, a complex assembly containing five small nuclear ribonucleoprotein (snRNP) particles. Human p14, a component of the spliceosomal U2 and U11/U12 snRNPs, has been shown to associate directly with the pre-mRNA branch adenosine early in spliceosome assembly and within the fully assembled spliceosome. Here we report the 2.5-A crystal structure of a complex containing p14 and a peptide derived from the p14-associated U2 snRNP component SF3b155. p14 contains an RNA recognition motif (RRM), the surface of which is largely occluded by a C-terminal alpha-helix and a portion of the SF3b155 peptide. An analysis of RNA.protein crosslinking to wild-type and mutant p14 shows that the branch adenosine directly interacts with a conserved aromatic within a pocket on the surface of the complex. This result, combined with a comparison of the structure with known RRMs and pseudoRRMs as well as model-building by using the electron cryomicroscopy structure of a spliceosomal U11/U12 di-snRNP, suggests that p14.SF3b155 presents a noncanonical surface for RNA recognition at the heart of the mammalian spliceosome. PMID:16432215

  9. Conserved structure of Snu13 from the highly reduced spliceosome of Cyanidioschyzon merolae.

    PubMed

    Black, C S; Garside, E L; MacMillan, A M; Rader, S D

    2016-04-01

    Structural and functional analysis of proteins involved in pre-mRNA splicing is challenging because of the complexity of the splicing machinery, known as the spliceosome. Bioinformatic, proteomic, and biochemical analyses have identified a minimal spliceosome in the red alga Cyanidioschyzon merolae. This spliceosome consists of only 40 core proteins, compared to ∼ 70 in S. cerevisiae (yeast) and ∼ 150 in humans. We report the X-ray crystallographic analysis of C. merolae Snu13 (CmSnu13), a key component of the assembling spliceosome, and present evidence for conservation of Snu13 function in this algal splicing pathway. The near identity of CmSnu13's three-dimensional structure to yeast and human Snu13 suggests that C. merolae should be an excellent model system for investigating the structure and function of the conserved core of the spliceosome. PMID:26833716

  10. A review of craniofacial disorders caused by spliceosomal defects.

    PubMed

    Lehalle, D; Wieczorek, D; Zechi-Ceide, R M; Passos-Bueno, M R; Lyonnet, S; Amiel, J; Gordon, C T

    2015-11-01

    The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNA transcripts. Mutations in EFTUD2, encoding a component of the major spliceosome, have recently been identified as the cause of mandibulofacial dysostosis, Guion-Almeida type (MFDGA), characterized by mandibulofacial dysostosis, microcephaly, external ear malformations and intellectual disability. Mutations in several other genes involved in spliceosomal function or linked aspects of mRNA processing have also recently been identified in human disorders with specific craniofacial malformations: SF3B4 in Nager syndrome, an acrofacial dysostosis (AFD); SNRPB in cerebrocostomandibular syndrome, characterized by Robin sequence and rib defects; EIF4A3 in the AFD Richieri-Costa-Pereira syndrome, characterized by Robin sequence, median mandibular cleft and limb defects; and TXNL4A in Burn-McKeown syndrome, involving specific craniofacial dysmorphisms. Here, we review phenotypic and molecular aspects of these syndromes. Given the apparent sensitivity of craniofacial development to defects in mRNA processing, it is possible that mutations in other proteins involved in spliceosomal function will emerge in the future as causative for related human disorders. PMID:25865758

  11. Nuclear cyclophilins affect spliceosome assembly and function in vitro

    PubMed Central

    Adams, B.M.; Coates, Miranda N.; Jackson, S. RaElle; Jurica, Melissa S.; Davis, Tara L.

    2015-01-01

    Cyclophilins are ubiquitously expressed proteins that bind to prolines and can catalyse cis/trans isomerization of proline residues. There are 17 annotated members of the cyclophilin family in humans, ubiquitously expressed and localized variously to the cytoplasm, nucleus or mitochondria. Surprisingly, all eight of the nuclear localized cyclophilins are found associated with spliceosomal complexes. However, their particular functions within this context are unknown. We have therefore adapted three established assays for in vitro pre-mRNA splicing to probe the functional roles of nuclear cyclophilins in the context of the human spliceosome. We find that four of the eight spliceosom-associated cyclophilins exert strong effects on splicing in vitro. These effects are dose-dependent and, remarkably, uniquely characteristic of each cyclophilin. Using both qualitative and quantitative means, we show that at least half of the nuclear cyclophilins can act as regulatory factors of spliceosome function in vitro. The present work provides the first quantifiable evidence that nuclear cyclophilins are splicing factors and provides a novel approach for future work into small molecule-based modulation of pre-mRNA splicing. PMID:25967372

  12. Expression of a human U1 RNA gene introduced into mouse cells via bovine papillomavirus DNA vectors.

    PubMed Central

    Schenborn, E T; Lund, E; Mitchen, J L; Dahlberg, J E

    1985-01-01

    We introduced a gene for human U1 small nuclear RNA, HU1-1, into mouse C127 cells via bovine papillomavirus (BPV) vectors. After transfection, up to 15% of the total U1 RNA in transformed cells was encoded by the introduced human genes. High levels of expression of the human gene were observed when the recombinant viral DNAs were maintained either as plasmids or after integration into high-molecular-weight DNA. As few as 400 and 35 base pairs of 5' and 3' flanking region sequences, respectively, were sufficient for transcription of human U1 RNA, and no increase in the level of expression was observed with HU1-1 DNA containing several kilobases of flanking region sequences. Several of the transformed cell lines contained the recombinant BPV DNA apparently integrated into the host genome. Integration or rearrangement or both of the U1-BPV DNA was promoted when the HU1-1 gene was positioned at the BamHI site downstream of the BPV transforming region. At least two variants of the U1-BPV DNAs were able to cause morphological transformation of cells despite the fact that these DNAs lacked a BPV transcriptional enhancer element. Images PMID:2412107

  13. Purification of Drosophila snRNPs and characterization of two populations of functional U1 particles.

    PubMed Central

    Labourier, E; Rio, D C

    2001-01-01

    U1 snRNP is required at an early stage during assembly of the spliceosome, the dynamic ribonucleoprotein (RNP) complex that performs nuclear pre-mRNA splicing. Here, we report the purification of U1 snRNP particles from Drosophila nuclear extracts and the characterization of their biochemical properties, polypeptide contents, and splicing activities. On the basis of their antigenicity, apparent molecular weight, and by peptide sequencing, the Drosophila 70K, SNF, B, U1-C, D1, D2, D3, E, F, and G proteins are shown to be integral components of these particles. Sequence database searches revealed that both the U1-specific and the Sm proteins are extensively conserved between human and Drosophila snRNPs. Furthermore, both species possess a conserved intrinsic U1-associated kinase activity with identical substrate specificity in vitro. Finally, our results demonstrate that a second type of functional U1 particle, completely lacking the U1/U2-specific protein SNF and the associated protein kinase activity, can be isolated from cultured Kc cell or Canton S embryonic nuclear extracts. This work describes the first characterization of a purified Drosophila snRNP particle and reinforces the view that their activity and composition, with the exception of the atypical bifunctional U1-A/U2-B" SNF protein, are highly conserved in metazoans. PMID:11333025

  14. Re-refinement of the spliceosomal U4 snRNP core-domain structure

    PubMed Central

    Li, Jade; Leung, Adelaine K.; Kondo, Yasushi; Oubridge, Chris; Nagai, Kiyoshi

    2016-01-01

    The core domain of small nuclear ribonucleoprotein (snRNP), comprised of a ring of seven paralogous proteins bound around a single-stranded RNA sequence, functions as the assembly nucleus in the maturation of U1, U2, U4 and U5 spliceosomal snRNPs. The structure of the human U4 snRNP core domain was initially solved at 3.6 Å resolution by experimental phasing using data with tetartohedral twinning. Molecular replacement from this model followed by density modification using untwinned data recently led to a structure of the minimal U1 snRNP at 3.3 Å resolution. With the latter structure providing a search model for molecular replacement, the U4 core-domain structure has now been re-refined. The U4 Sm site-sequence AAUUUUU has been shown to bind to the seven Sm proteins SmF–SmE–SmG–SmD3–SmB–SmD1–SmD2 in an identical manner as the U1 Sm-site sequence AAUUUGU, except in SmD1 where the bound U replaces G. The progression from the initial to the re-refined structure exemplifies a tortuous route to accuracy: where well diffracting crystals of complex assemblies are initially unavailable, the early model errors are rectified by exploiting preliminary interpretations in further experiments involving homologous structures. New insights are obtained from the more accurate model. PMID:26894541

  15. Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis.

    PubMed Central

    Jurica, Melissa S; Licklider, Lawrence J; Gygi, Steven R; Grigorieff, Nikolaus; Moore, Melissa J

    2002-01-01

    We describe characterization of spliceosomes affinity purified under native conditions. These spliceosomes consist largely of C complex containing splicing intermediates. After C complex assembly on an MS2 affinity-tagged pre-mRNA substrate containing a 3' splice site mutation, followed by RNase H digestion of earlier complexes, spliceosomes were purified by size exclusion and affinity selection. This protocol yielded 40S C complexes in sufficient quantities to visualize in negative stain by electron microscopy. Complexes purified in this way contain U2, U5, and U6 snRNAs, but very little U1 or U4 snRNA. Analysis by tandem mass spectrometry confirmed the presence of core snRNP proteins (SM and LSM), U2 and U5 snRNP-specific proteins, and the second step factors Prp16, Prp17, Slu7, and Prp22. In contrast, proteins specific to earlier splicing complexes, such as U2AF and U1 snRNP components, were not detected in C complex, but were present in similarly purified H complex. Images of these spliceosomes revealed single particles with dimensions of approximately 270 x 240 A that assort into well-defined classes. These images represent an important first step toward attaining a comprehensive three-dimensional understanding of pre-mRNA splicing. PMID:11991638

  16. Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5′-cap structure

    PubMed Central

    Simoes-Barbosa, Augusto; Meloni, Dionigia; Wohlschlegel, James A.; Konarska, Maria M.; Johnson, Patricia J.

    2008-01-01

    Few genes in the divergent eukaryote Trichomonas vaginalis have introns, despite the unusually large gene repertoire of this human-infective parasite. These introns are characterized by extended conserved regulatory motifs at the 5′ and 3′ boundaries, a feature shared with another divergent eukaryote, Giardia lamblia, but not with metazoan introns. This unusual characteristic of T. vaginalis introns led us to examine spliceosomal small nuclear RNAs (snRNAs) predicted to mediate splicing reactions via interaction with intron motifs. Here we identify T. vaginalis U1, U2, U4, U5, and U6 snRNAs, present predictions of their secondary structures, and provide evidence for interaction between the U2/U6 snRNA complex and a T. vaginalis intron. Structural models predict that T. vaginalis snRNAs contain conserved sequences and motifs similar to those found in other examined eukaryotes. These data indicate that mechanisms of intron recognition as well as coordination of the two catalytic steps of splicing have been conserved throughout eukaryotic evolution. Unexpectedly, we found that T. vaginalis spliceosomal snRNAs lack the 5′ trimethylguanosine cap typical of snRNAs and appear to possess unmodified 5′ ends. Despite the lack of a cap structure, U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III. PMID:18596255

  17. Origin of Spliceosomal Introns and Alternative Splicing

    PubMed Central

    Irimia, Manuel; Roy, Scott William

    2014-01-01

    In this work we review the current knowledge on the prehistory, origins, and evolution of spliceosomal introns. First, we briefly outline the major features of the different types of introns, with particular emphasis on the nonspliceosomal self-splicing group II introns, which are widely thought to be the ancestors of spliceosomal introns. Next, we discuss the main scenarios proposed for the origin and proliferation of spliceosomal introns, an event intimately linked to eukaryogenesis. We then summarize the evidence that suggests that the last eukaryotic common ancestor (LECA) had remarkably high intron densities and many associated characteristics resembling modern intron-rich genomes. From this intron-rich LECA, the different eukaryotic lineages have taken very distinct evolutionary paths leading to profoundly diverged modern genome structures. Finally, we discuss the origins of alternative splicing and the qualitative differences in alternative splicing forms and functions across lineages. PMID:24890509

  18. Defining the orientation of the human U1A RBD1 on its UTR by tethered-EDTA(Fe) cleavage.

    PubMed Central

    Beck, D L; Stump, W T; Hall, K B

    1998-01-01

    The N-terminal RNA binding domain of the human U1A protein (RBD1) specifically binds an RNA hairpin of U1 snRNA as well as two internal loops in the 3' UTR of its own mRNA. Here, a single cysteine has been introduced into Loop 1 of RBD1, which is subsequently used to attach (EDTA-2-aminoethyl) 2-pyridyl disulfide-Fe3+ (EPD-Fe). This EDTA-Fe derivative is used to generate hydroxyl radicals to cleave the proximal RNA sugar-phosphate backbone in the RNA-RBD complexes. RBD1(K20C)-EPD-Fe cleaves the 5' strand of the RNA hairpin stem, centered four base pairs away from the base of the loop, and cleaves the UTR in two places, again centered on the 5' side of the fourth base pair from each internal loop. These data, extrapolated to the position of Lys 20 in RBD1, orient the two proteins bound to the UTR, and provide direct biochemical evidence for the proposed model of the RBD1:UTR complex. PMID:9510334

  19. The spliceosomal PRP19 complex of trypanosomes

    PubMed Central

    Ambrósio, Daniela L.; Badjatia, Nitika; Günzl, Arthur

    2015-01-01

    Summary In trypanosomes, mRNAs are processed by spliced leader (SL) trans splicing, in which a capped SL, derived from SL RNA, is spliced onto the 5′ end of each mRNA. This process is mediated by the spliceosome, a large and dynamic RNA-protein machinery consisting of small nuclear ribonucleoproteins (snRNPs) and non-snRNP proteins. Due to early evolutionary divergence, the amino acid sequences of trypanosome splicing factors exhibit limited similarity to those of their eukaryotic orthologs making their bioinformatic identification challenging. Most of the ~ 60 protein components that have been characterized thus far are snRNP proteins because, in contrast to individual snRNPs, purification of intact spliceosomes has not been achieved yet. Here, we characterize the non-snRNP PRP19 complex of Trypanosoma brucei. We identified a complex that contained the core subunits PRP19, CDC5, PRL1, and SPF27, as well as PRP17, SKIP and PPIL1. Three of these proteins were newly annotated. The PRP19 complex was associated primarily with the activated spliceosome and, accordingly, SPF27 silencing blocked the first splicing step. Interestingly, SPF27 silencing caused an accumulation of SL RNA with a hypomethylated cap that closely resembled the defect observed previously upon depletion of the cyclin-dependent kinase CRK9, indicating that both proteins may function in spliceosome activation. PMID:25524563

  20. Detailed close-ups and the big picture of spliceosomes

    PubMed Central

    Jurica, Melissa S.

    2008-01-01

    Summary The spliceosome is the huge macromolecular assembly responsible for the removal of introns from pre-mRNA transcripts. The size and complexity of this dynamic cellular machine dictates that structural analysis of the spliceosome is best served by a combination of techniques. Electron microscopy is providing a more global, albeit less detailed, view of spliceosome assemblies. X-ray crystallographers and NMR spectroscopists are steadily reporting more atomic resolution structures of individual spliceosome components and fragments. Increasingly, structures of these individual pieces in complex with binding partners are yielding insights into the interfaces that hold the entire spliceosome assembly together. Although the information arising from the various structural studies of splicing machinery has not yet fully converged into a complete model, we can expect that a detailed understanding of spliceosome structure will arise at the juncture of structural and computational modeling methods. PMID:18550358

  1. The architecture of the spliceosomal U4/U6.U5 tri-snRNP

    PubMed Central

    Nguyen, Thi Hoang Duong; Galej, Wojciech P.; Bai, Xiao-chen; Savva, Christos G.; Newman, Andrew J.; Scheres, Sjors H. W.; Nagai, Kiyoshi

    2015-01-01

    U4/U6.U5 tri-snRNP is a 1.5 MDa pre-assembled spliceosomal complex comprising U5 snRNA, extensively base-paired U4/U6 snRNAs and >30 proteins, including the key components Prp8, Brr2 and Snu114. The tri-snRNP combines with a pre-mRNA substrate bound to U1 and U2 snRNPs and transforms into a catalytically active spliceosome following extensive compositional and conformational changes triggered by unwinding of the U4/U6 snRNAs. CryoEM single-particle reconstruction of yeast tri-snRNP at 5.9Å resolution reveals the essentially complete organization of its RNA and protein components. The single-stranded region of U4 snRNA between its 3′-stem-loop and the U4/U6 snRNA stem I is loaded into the Brr2 helicase active site ready for unwinding. Snu114 and the N-terminal domain of Prp8 position U5 snRNA to insert its Loop I, which aligns the exons for splicing, into the Prp8 active site cavity. The structure provides crucial insights into the activation process and the active site of the spliceosome. PMID:26106855

  2. Co-evolution of SNF spliceosomal proteins with their RNA targets in trans-splicing nematodes.

    PubMed

    Strange, Rex Meade; Russelburg, L Peyton; Delaney, Kimberly J

    2016-08-01

    Although the mechanism of pre-mRNA splicing has been well characterized, the evolution of spliceosomal proteins is poorly understood. The U1A/U2B″/SNF family (hereafter referred to as the SNF family) of RNA binding spliceosomal proteins participates in both the U1 and U2 small interacting nuclear ribonucleoproteins (snRNPs). The highly constrained nature of this system has inhibited an analysis of co-evolutionary trends between the proteins and their RNA binding targets. Here we report accelerated sequence evolution in the SNF protein family in Phylum Nematoda, which has allowed an analysis of protein:RNA co-evolution. In a comparison of SNF genes from ecdysozoan species, we found a correlation between trans-splicing species (nematodes) and increased phylogenetic branch lengths of the SNF protein family, with respect to their sister clade Arthropoda. In particular, we found that nematodes (~70-80 % of pre-mRNAs are trans-spliced) have experienced higher rates of SNF sequence evolution than arthropods (predominantly cis-spliced) at both the nucleotide and amino acid levels. Interestingly, this increased evolutionary rate correlates with the reliance on trans-splicing by nematodes, which would alter the role of the SNF family of spliceosomal proteins. We mapped amino acid substitutions to functionally important regions of the SNF protein, specifically to sites that are predicted to disrupt protein:RNA and protein:protein interactions. Finally, we investigated SNF's RNA targets: the U1 and U2 snRNAs. Both are more divergent in nematodes than arthropods, suggesting the RNAs have co-evolved with SNF in order to maintain the necessarily high affinity interaction that has been characterized in other species. PMID:27450547

  3. A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity.

    PubMed

    Boesler, Carsten; Rigo, Norbert; Anokhina, Maria M; Tauchert, Marcel J; Agafonov, Dmitry E; Kastner, Berthold; Urlaub, Henning; Ficner, Ralf; Will, Cindy L; Lührmann, Reinhard

    2016-01-01

    The precise role of the spliceosomal DEAD-box protein Prp28 in higher eukaryotes remains unclear. We show that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity. Complexes formed in the presence of ATPase-deficient hPrp28 represent a novel assembly intermediate, the pre-B complex, that contains U1, U2 and loosely associated tri-snRNP and is stalled before disruption of the U1/5'ss base pairing interaction, consistent with a role for hPrp28 in the latter. Pre-B and B complexes differ structurally, indicating that stable tri-snRNP integration is accompanied by substantial rearrangements in the spliceosome. Disruption of the U1/5'ss interaction alone is not sufficient to bypass the block by ATPase-deficient hPrp28, suggesting hPrp28 has an additional function at this stage of splicing. Our data provide new insights into the function of Prp28 in higher eukaryotes, and the requirements for stable tri-snRNP binding during B complex formation. PMID:27377154

  4. A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity

    PubMed Central

    Boesler, Carsten; Rigo, Norbert; Anokhina, Maria M.; Tauchert, Marcel J.; Agafonov, Dmitry E.; Kastner, Berthold; Urlaub, Henning; Ficner, Ralf; Will, Cindy L.; Lührmann, Reinhard

    2016-01-01

    The precise role of the spliceosomal DEAD-box protein Prp28 in higher eukaryotes remains unclear. We show that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity. Complexes formed in the presence of ATPase-deficient hPrp28 represent a novel assembly intermediate, the pre-B complex, that contains U1, U2 and loosely associated tri-snRNP and is stalled before disruption of the U1/5′ss base pairing interaction, consistent with a role for hPrp28 in the latter. Pre-B and B complexes differ structurally, indicating that stable tri-snRNP integration is accompanied by substantial rearrangements in the spliceosome. Disruption of the U1/5′ss interaction alone is not sufficient to bypass the block by ATPase-deficient hPrp28, suggesting hPrp28 has an additional function at this stage of splicing. Our data provide new insights into the function of Prp28 in higher eukaryotes, and the requirements for stable tri-snRNP binding during B complex formation. PMID:27377154

  5. Tumor suppressor microRNAs are downregulated in myelodysplastic syndrome with spliceosome mutations

    PubMed Central

    Aslan, Derya; Garde, Christian; Nygaard, Mette Katrine; Helbo, Alexandra Søgaard; Dimopoulos, Konstantinos; Hansen, Jakob Werner; Severinsen, Marianne Tang; Treppendahl, Marianne Bach; Sjø, Lene Dissing; Grønbæk, Kirsten; Kristensen, Lasse Sommer

    2016-01-01

    Spliceosome mutations are frequently observed in patients with myelodysplastic syndromes (MDS). However, it is largely unknown how these mutations contribute to the disease. MicroRNAs (miRNAs) are small noncoding RNAs, which have been implicated in most human cancers due to their role in post transcriptional gene regulation. The aim of this study was to analyze the impact of spliceosome mutations on the expression of miRNAs in a cohort of 34 MDS patients. In total, the expression of 76 miRNAs, including mirtrons and splice site overlapping miRNAs, was accurately quantified using reverse transcriptase quantitative PCR. The majority of the studied miRNAs have previously been implicated in MDS. Stably expressed miRNA genes for normalization of the data were identified using GeNorm and NormFinder algorithms. High-resolution melting assays covering all mutational hotspots within SF3B1, SRSF2, and U2AF1 (U2AF35) were developed, and all detected mutations were confirmed by Sanger sequencing. Overall, canonical miRNAs were downregulated in spliceosome mutated samples compared to wild-type (P = 0.002), and samples from spliceosome mutated patients clustered together in hierarchical cluster analyses. Among the most downregulated miRNAs were several tumor-suppressor miRNAs, including several let-7 family members, miR-423, and miR-103a. Finally, we observed that the predicted targets of the most downregulated miRNAs were involved in apoptosis, hematopoiesis, and acute myeloid leukemia among other cancer- and metabolic pathways. Our data indicate that spliceosome mutations may play an important role in MDS pathophysiology by affecting the expression of tumor suppressor miRNA genes involved in the development and progression of MDS. PMID:26848861

  6. Graded requirement for the spliceosome in cell cycle progression

    PubMed Central

    Karamysheva, Zemfira; Díaz-Martínez, Laura A; Warrington, Ross; Yu, Hongtao

    2015-01-01

    Genome stability is ensured by multiple surveillance mechanisms that monitor the duplication, segregation, and integrity of the genome throughout the cell cycle. Depletion of components of the spliceosome, a macromolecular machine essential for mRNA maturation and gene expression, has been associated with increased DNA damage and cell cycle defects. However, the specific role for the spliceosome in these processes has remained elusive, as different cell cycle defects have been reported depending on the specific spliceosome subunit depleted. Through a detailed cell cycle analysis after spliceosome depletion, we demonstrate that the spliceosome is required for progression through multiple phases of the cell cycle. Strikingly, the specific cell cycle phenotype observed after spliceosome depletion correlates with the extent of depletion. Partial depletion of a core spliceosome component results in defects at later stages of the cell cycle (G2 and mitosis), whereas a more complete depletion of the same component elicits an early cell cycle arrest in G1. We propose a quantitative model in which different functional dosages of the spliceosome are required for different cell cycle transitions. PMID:25892155

  7. The human U1-70K snRNP protein: cDNA cloning, chromosomal localization, expression, alternative splicing and RNA-binding.

    PubMed Central

    Spritz, R A; Strunk, K; Surowy, C S; Hoch, S O; Barton, D E; Francke, U

    1987-01-01

    We have isolated and sequenced cDNA clones encoding the human U1-70K snRNP protein, and have mapped this locus (U1AP1) to human chromosome 19. The gene produces two size classes of RNA, a major 1.7-kb RNA and a minor 3.9-kb RNA. The 1.7-kb species appears to be the functional mRNA; the role of the 3.9-kb RNA, which extends further in the 5' direction, is unclear. The actual size of the hU1-70K protein is probably 52 kd, rather than 70 kd. The protein contains three regions similar to known nucleic acid-binding proteins, and it binds RNA in an in vitro assay. Comparison of the cDNA sequences indicates that there are multiple subclasses of mRNA that arise by alternative pre-mRNA splicing of at least four alternative exon segments. This suggests that multiple forms of the hU1-70K protein may exist, possibly with different functions in vivo. Images PMID:2447561

  8. Expression of U1 small nuclear ribonucleoprotein 70K antisense transcript using APETALA3 promoter suppresses the development of sepals and petals.

    PubMed

    Golovkin, Maxim; Reddy, Anireddy S N

    2003-08-01

    U1 small nuclear ribonucleoprotein (snRNP)-70K (U1-70K), a U1 snRNP-specific protein, is involved in the early stages of spliceosome formation. In non-plant systems, it is involved in constitutive and alternative splicing. It has been shown that U1snRNP is dispensable for in vitro splicing of some animal pre-mRNAs, and inactivation of U1-70K in yeast (Saccharomyces cerevisiae) is not lethal. As in yeast and humans (Homo sapiens), plant U1-70K is coded by a single gene. In this study, we blocked the expression of Arabidopsis U1-70K in petals and stamens by expressing U1-70K antisense transcript using the AP3 (APETALA3) promoter specific to these floral organs. Flowers of transgenic Arabidopsis plants expressing U1-70K antisense transcript showed partially developed stamens and petals that are arrested at different stages of development. In some transgenic lines, flowers have rudimentary petals and stamens and are male sterile. The severity of the phenotype is correlated with the level of the antisense transcript. Molecular analysis of transgenic plants has confirmed that the observed phenotype is not due to disruption of whorl-specific homeotic genes, AP3 or PISTILLATA, responsible for petal and stamen development. The AP3 transcript was not detected in transgenic flowers with severe phenotype. Flowers of Arabidopsis plants transformed with a reporter gene driven by the same promoter showed no abnormalities. These results show that U1-70K is necessary for the development of sepals and petals and is an essential gene in plants. PMID:12913145

  9. SNEV is an evolutionarily conserved splicing factor whose oligomerization is necessary for spliceosome assembly

    PubMed Central

    Grillari, Johannes; Ajuh, Paul; Stadler, Guido; Löscher, Marlies; Voglauer, Regina; Ernst, Wolfgang; Chusainow, Janet; Eisenhaber, Frank; Pokar, Marion; Fortschegger, Klaus; Grey, Martin; Lamond, Angus I.; Katinger, Hermann

    2005-01-01

    We have isolated the human protein SNEV as downregulated in replicatively senescent cells. Sequence homology to the yeast splicing factor Prp19 suggested that SNEV might be the orthologue of Prp19 and therefore might also be involved in pre-mRNA splicing. We have used various approaches including gene complementation studies in yeast using a temperature sensitive mutant with a pleiotropic phenotype and SNEV immunodepletion from human HeLa nuclear extracts to determine its function. A human–yeast chimera was indeed capable of restoring the wild-type phenotype of the yeast mutant strain. In addition, immunodepletion of SNEV from human nuclear extracts resulted in a decrease of in vitro pre-mRNA splicing efficiency. Furthermore, as part of our analysis of protein–protein interactions within the CDC5L complex, we found that SNEV interacts with itself. The self-interaction domain was mapped to amino acids 56–74 in the protein's sequence and synthetic peptides derived from this region inhibit in vitro splicing by surprisingly interfering with spliceosome formation and stability. These results indicate that SNEV is the human orthologue of yeast PRP19, functions in splicing and that homo-oligomerization of SNEV in HeLa nuclear extract is essential for spliceosome assembly and that it might also be important for spliceosome stability. PMID:16332694

  10. Autoimmune response to U1 small nuclear ribonucleoprotein (U1 snRNP) associated with cytomegalovirus infection.

    PubMed

    Newkirk, M M; van Venrooij, W J; Marshall, G S

    2001-01-01

    The induction of autoantibodies to U1 small nuclear ribonucleoprotein (U1 snRNP) complexes is not well understood. We present evidence that healthy individuals with cytomegalovirus (CMV) infection have an increased frequency and quantity of antibodies to ribonucleoprotein, directed primarily against the U1-70k protein. A significant association between the presence of antibodies to CMV and antibodies to the total RNP targeted by the immune response to the spliceosome (to both the Sm and RNP; Sm/RNP) was found for patients with systemic lupus erythematosus (SLE) but not those with mixed connective-tissue disease. CMV thus may play a role in inducing autoimmune responses in a subset of patients with systemic lupus erythematosus. PMID:11438044

  11. The Spliceosome: The Ultimate RNA Chaperone and Sculptor.

    PubMed

    Papasaikas, Panagiotis; Valcárcel, Juan

    2016-01-01

    The spliceosome, one of the most complex machineries of eukaryotic cells, removes intronic sequences from primary transcripts to generate functional messenger and long noncoding RNAs (lncRNA). Genetic, biochemical, and structural data reveal that the spliceosome is an RNA-based enzyme. Striking mechanistic and structural similarities strongly argue that pre-mRNA introns originated from self-catalytic group II ribozymes. However, in the spliceosome, protein components organize and activate the catalytic-site RNAs, and recognize and pair together splice sites at intron boundaries. The spliceosome is a dynamic, reversible, and flexible machine that chaperones small nuclear (sn) RNAs and a variety of pre-mRNA sequences into conformations that enable intron removal. This malleability likely contributes to the regulation of alternative splicing, a prevalent process contributing to cell differentiation, homeostasis, and disease. PMID:26682498

  12. Haploinsufficiency of a Spliceosomal GTPase Encoded by EFTUD2 Causes Mandibulofacial Dysostosis with Microcephaly

    PubMed Central

    Lines, Matthew A.; Huang, Lijia; Schwartzentruber, Jeremy; Douglas, Stuart L.; Lynch, Danielle C.; Beaulieu, Chandree; Guion-Almeida, Maria Leine; Zechi-Ceide, Roseli Maria; Gener, Blanca; Gillessen-Kaesbach, Gabriele; Nava, Caroline; Baujat, Geneviève; Horn, Denise; Kini, Usha; Caliebe, Almuth; Alanay, Yasemin; Utine, Gulen Eda; Lev, Dorit; Kohlhase, Jürgen; Grix, Arthur W.; Lohmann, Dietmar R.; Hehr, Ute; Böhm, Detlef; Majewski, Jacek; Bulman, Dennis E.; Wieczorek, Dagmar; Boycott, Kym M.

    2012-01-01

    Mandibulofacial dysostosis with microcephaly (MFDM) is a rare sporadic syndrome comprising craniofacial malformations, microcephaly, developmental delay, and a recognizable dysmorphic appearance. Major sequelae, including choanal atresia, sensorineural hearing loss, and cleft palate, each occur in a significant proportion of affected individuals. We present detailed clinical findings in 12 unrelated individuals with MFDM; these 12 individuals compose the largest reported cohort to date. To define the etiology of MFDM, we employed whole-exome sequencing of four unrelated affected individuals and identified heterozygous mutations or deletions of EFTUD2 in all four. Validation studies of eight additional individuals with MFDM demonstrated causative EFTUD2 mutations in all affected individuals tested. A range of EFTUD2-mutation types, including null alleles and frameshifts, is seen in MFDM, consistent with haploinsufficiency; segregation is de novo in all cases assessed to date. U5-116kD, the protein encoded by EFTUD2, is a highly conserved spliceosomal GTPase with a central regulatory role in catalytic splicing and post-splicing-complex disassembly. MFDM is the first multiple-malformation syndrome attributed to a defect of the major spliceosome. Our findings significantly extend the range of reported spliceosomal phenotypes in humans and pave the way for further investigation in related conditions such as Treacher Collins syndrome. PMID:22305528

  13. U1 RNA Induces Innate Immunity Signaling

    PubMed Central

    Hoffman, Robert W.; Gazitt, Tal; Foecking, Mark F.; Ortmann, Robert A.; Misfeldt, Michael; Jorgenson, Rebecca; Young, Steven L.; Greidinger, Eric L.

    2006-01-01

    Objective The U1–70-kd RNP is a prominent target of autoimmunity in connective tissue diseases. In this study, we explored whether its endogenous ligand, U1 RNA, mediates a proimmune signal and may be immunogenic. Methods We assayed the proliferation of control and MyD88-knockout splenocytes in response to in vitro–synthesized U1 RNA, and measured interleukin-6 (IL-6) and IL-8 secretion induced by U1 RNA in a human cell line competent for signaling through Toll-like receptor 3 (TLR-3) and TLR-5. Results Treatment with U1 RNA or with poly(I-C), a known agonist of TLR-3, induced approximately twice as much control splenocyte proliferation as did treatment with RNase-digested U1 RNA. Proliferation in response to either poly(I-C) or U1 RNA by MyD88-knockout splenocytes was similarly attenuated. Similar to poly(I-C), U1 RNA induced significant secretion of both IL-6 and IL-8 from a TLR-3–expressing human cell line; in contrast, the TLR-5 agonist flagellin induced predominantly IL-8 secretion. Pretreatment of U1 RNA with RNase abolished IL-6 and IL-8 secretion. Conclusion U1 RNA is capable of inducing manifestations consistent with TLR-3 activation. The ability of U1 RNA (which has a substantial double-stranded secondary structure) to activate TLR-3 may contribute to the immunogenicity of the U1–70-kd autoantigen. Stimulation of innate immunity by native RNA molecules with a double-stranded secondary structure may help explain the high prevalence of autoimmunity to RNA binding proteins. PMID:15457457

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    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

  16. U1A Complex

    SciTech Connect

    2014-10-28

    Some of the most sophisticated experiments in the stockpile stewardship program are conducted in an environmentally safe manner, nearly 1000 feet below the ground at the site. The U1a complex a sprawling underground laboratory and tunnel complex is home to a number of unique capabilities.

  17. U1A Complex

    ScienceCinema

    None

    2015-01-09

    Some of the most sophisticated experiments in the stockpile stewardship program are conducted in an environmentally safe manner, nearly 1000 feet below the ground at the site. The U1a complex a sprawling underground laboratory and tunnel complex is home to a number of unique capabilities.

  18. New insights into the spliceosome by single molecule fluorescence microscopy.

    PubMed

    Hoskins, Aaron A; Gelles, Jeff; Moore, Melissa J

    2011-12-01

    Splicing is an essential eukaryotic process in which introns are excised from precursors to messenger RNAs and exons ligated together. This reaction is catalyzed by a multi-MegaDalton machine called the spliceosome, composed of 5 small nuclear RNAs (snRNAs) and a core set of ∼100 proteins minimally required for activity. Because of the spliceosome's size, its low abundance in cellular extracts, and its highly dynamic assembly pathway, analysis of the kinetics of splicing and the conformational rearrangements occurring during spliceosome assembly and disassembly has proven extraordinarily challenging. Here, we review recent progress in combining chemical biology methodologies with single molecule fluorescence techniques to provide a window into splicing in real time. These methods complement ensemble measurements of splicing in vivo and in vitro to facilitate kinetic dissection of pre-mRNA splicing. PMID:22057211

  19. Analysis of spliceosomal proteins in Trypanosomatids reveals novel functions in mRNA processing.

    PubMed

    Tkacz, Itai Dov; Gupta, Sachin Kumar; Volkov, Vadim; Romano, Mali; Haham, Tomer; Tulinski, Pawel; Lebenthal, Ilana; Michaeli, Shulamit

    2010-09-01

    In trypanosomatids, all mRNAs are processed via trans-splicing, although cis-splicing also occurs. In trans-splicing, a common small exon, the spliced leader (SL), which is derived from a small SL RNA species, is added to all mRNAs. Sm and Lsm proteins are core proteins that bind to U snRNAs and are essential for both these splicing processes. In this study, SmD3- and Lsm3-associated complexes were purified to homogeneity from Leishmania tarentolae. The purified complexes were analyzed by mass spectrometry, and 54 and 39 proteins were purified from SmD3 and Lsm complexes, respectively. Interestingly, among the proteins purified from Lsm3, no mRNA degradation factors were detected, as in Lsm complexes from other eukaryotes. The U1A complex was purified and mass spectrometry analysis identified, in addition to U1 small nuclear ribonucleoprotein (snRNP) proteins, additional co-purified proteins, including the polyadenylation factor CPSF73. Defects observed in cells silenced for U1 snRNP proteins suggest that the U1 snRNP functions exclusively in cis-splicing, although U1A also participates in polyadenylation and affects trans-splicing. The study characterized several trypanosome-specific nuclear factors involved in snRNP biogenesis, whose function was elucidated in Trypanosoma brucei. Conserved factors, such as PRP19, which functions at the heart of every cis-spliceosome, also affect SL RNA modification; GEMIN2, a protein associated with SMN (survival of motor neurons) and implicated in selective association of U snRNA with core Sm proteins in trypanosomes, is a master regulator of snRNP assembly. This study demonstrates the existence of trypanosomatid-specific splicing factors but also that conserved snRNP proteins possess trypanosome-specific functions. PMID:20592024

  20. Origin and evolution of spliceosomal introns

    PubMed Central

    2012-01-01

    Evolution of exon-intron structure of eukaryotic genes has been a matter of long-standing, intensive debate. The introns-early concept, later rebranded ‘introns first’ held 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. The introns-late concept held that introns emerged only in eukaryotes and new introns have been accumulating continuously throughout eukaryotic evolution. Analysis of orthologous genes from completely sequenced eukaryotic genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists, suggesting that many ancestral introns have persisted since the last eukaryotic common ancestor (LECA). Reconstructions of intron gain and loss using the growing collection of genomes of diverse eukaryotes and increasingly advanced probabilistic models convincingly show that the LECA and the ancestors of each eukaryotic supergroup had intron-rich genes, with intron densities comparable to those in the most intron-rich modern genomes such as those of vertebrates. The subsequent evolution in most lineages of eukaryotes involved primarily loss of introns, with only a few episodes of substantial intron gain that might have accompanied major evolutionary innovations such as the origin of metazoa. The original invasion of self-splicing Group II introns, presumably originating from the mitochondrial endosymbiont, into the genome of the emerging eukaryote might have been a key factor of eukaryogenesis that in particular triggered the origin of endomembranes and the nucleus. Conversely, splicing errors gave rise to alternative splicing, a major contribution to the biological complexity of multicellular eukaryotes. There is no indication that any prokaryote has ever possessed a spliceosome

  1. Use of the U1A Protein to Facilitate Crystallization and Structure Determination of Large RNAs.

    PubMed

    Ferré-D'Amaré, Adrian R

    2016-01-01

    The preparation of well-ordered crystals of RNAs with complex three-dimensional architecture can be facilitated by engineering a binding site for the spliceosomal protein U1A into a functionally and structurally dispensable stem-loop of the RNA of interest. Once suitable crystals are obtained, the U1A protein, of known structure, can be employed to facilitate preparation of heavy atom or anomalously scattering atom derivatives, or as a source of partial model phases for the molecular replacement method. Here, we describe the methods for making U1A preparations suitable for cocrystallization with RNA. As an example, the cocrystallization of the tetracycline aptamer with U1A is also described. PMID:26227038

  2. Rhodamine 123 phototoxicity in laser-irradiated MGH-U1 human carcinoma cells studied in vitro by electron microscopy and confocal laser scanning microscopy

    SciTech Connect

    Shea, C.R.; Sherwood, M.E.; Flotte, T.J.; Chen, N.; Scholz, M.; Hasan, T. )

    1990-07-01

    Rhodamine 123 (R123) is a permeant, cationic, fluorescent dye that localizes preferentially within mitochondria of living carcinoma cells. MGH-U1 human bladder carcinoma cells incubated in vitro with 10 microM R123 for 30 min and then irradiated at 514.5 nm with an argon ion laser underwent selective, phototoxic injury to mitochondria. Ultrastructurally, treatment with R123 plus irradiation with 10 J/cm2 caused selective, progressive mitochondrial alterations consisting of disruption of cristae, vacuolization, swelling, increasing numbers of ring-shaped and angulated mitochondria at 4 to 8 h after irradiation, and obliteration of many mitochondria at 24 to 48 h. Confocal laser scanning microscopy after treatment with R123 plus irradiation with 10 to 30 J/cm2 demonstrated altered uptake and localization of subsequently administered R123, accompanied by striking mitochondrial fragmentation. Irradiation caused a dose-dependent depletion of extractable R123, due to a photosensitized efflux that began immediately and progressed by 4 h after irradiation with 10 to 30 J/cm2; further uptake after reincubation in the presence of R123 was also quantitatively impaired in cells previously irradiated with 30 J/cm2.

  3. U1h Superstructure

    SciTech Connect

    Glen Sykes

    2000-11-01

    The U1H Shaft Project is a design build subcontract to supply the U. S. Department of Energy (DOE) a 1,045 ft. deep, 20 ft. diameter, concrete lined shaft for unspecified purposes. The subcontract awarded to Atkinson Construction by Bechtel Nevada to design and construct the shaft for the DOE has been split into phases with portions of the work being released as dictated by available funding. The first portion released included the design for the shaft, permanent hoist, headframe, and collar arrangement. The second release consisted of constructing the shaft collar to a depth of 110 ft., the service entry, utility trenches, and installation of the temporary sinking plant. The temporary sinking plant included the installation of the sinking headframe, the sinking hoist, two deck winches, the shaft form, the sinking work deck, and temporary utilities required to sink the shaft. Both the design and collar construction were completed on schedule. The third release consisted of excavating and lining the shaft to the station depth of approximately 950 feet. Work is currently proceeding on this production sinking phase. At a depth of approximately 600 feet, Atkinson has surpassed production expectation and is more than 3 months ahead of schedule. Atkinson has employed the use of a Bobcat 331 excavator as the primary means of excavation. the shaft is being excavated entirely in an alluvial deposit with varying degrees of calcium carbonate cementation. Several more work packages are expected to be released in the near future. The remaining work packages include, construction of the shaft station a depth of 975 ft. and construction of the shaft sump to a depth of 1,045 ft., installation of the loading pocket and station steel and equipment, installation of the shaft steel and guides, installation of the shaft utilities, and installation of the permanent headframe, hoist, collar utilities, and facilities.

  4. Vought XF3U-1

    NASA Technical Reports Server (NTRS)

    1933-01-01

    Vought XF3U-1: The Vought XF3U-1 was tested in Langley's 30 x 60 Full Scale Tunnel in 1933. The XF3U-1 was built in response to a Navy specification for a two-seat fighter. This aircraft was later used as an engine testbed. The design was revised for a scout bomber requirement and in this form saw service as the SBU.

  5. Vought XSB3U-1

    NASA Technical Reports Server (NTRS)

    1938-01-01

    Vought XSB3U-1: This Navy biplane scout bomber was the first biplane built by Vought with retractable gear. Note that his XSB3U-1 has a test boom off the left upper wing. The bomber was at Langley for NACA's investigation of tail loads.

  6. U1 snDNA clusters in grasshoppers: chromosomal dynamics and genomic organization

    PubMed Central

    Anjos, A; Ruiz-Ruano, F J; Camacho, J P M; Loreto, V; Cabrero, J; de Souza, M J; Cabral-de-Mello, D C

    2015-01-01

    The spliceosome, constituted by a protein set associated with small nuclear RNA (snRNA), is responsible for mRNA maturation through intron removal. Among snRNA genes, U1 is generally a conserved repetitive sequence. To unveil the chromosomal/genomic dynamics of this multigene family in grasshoppers, we mapped U1 genes by fluorescence in situ hybridization in 70 species belonging to the families Proscopiidae, Pyrgomorphidae, Ommexechidae, Romaleidae and Acrididae. Evident clusters were observed in all species, indicating that, at least, some U1 repeats are tandemly arrayed. High conservation was observed in the first four families, with most species carrying a single U1 cluster, frequently located in the third or fourth longest autosome. By contrast, extensive variation was observed among Acrididae, from a single chromosome pair carrying U1 to all chromosome pairs carrying it, with occasional occurrence of two or more clusters in the same chromosome. DNA sequence analysis in Eyprepocnemis plorans (species carrying U1 clusters on seven different chromosome pairs) and Locusta migratoria (carrying U1 in a single chromosome pair) supported the coexistence of functional and pseudogenic lineages. One of these pseudogenic lineages was truncated in the same nucleotide position in both species, suggesting that it was present in a common ancestor to both species. At least in E. plorans, this U1 snDNA pseudogenic lineage was associated with 5S rDNA and short interspersed elements (SINE)-like mobile elements. Given that we conclude in grasshoppers that the U1 snDNA had evolved under the birth-and-death model and that its intragenomic spread might be related with mobile elements. PMID:25248465

  7. U1 snDNA clusters in grasshoppers: chromosomal dynamics and genomic organization.

    PubMed

    Anjos, A; Ruiz-Ruano, F J; Camacho, J P M; Loreto, V; Cabrero, J; de Souza, M J; Cabral-de-Mello, D C

    2015-02-01

    The spliceosome, constituted by a protein set associated with small nuclear RNA (snRNA), is responsible for mRNA maturation through intron removal. Among snRNA genes, U1 is generally a conserved repetitive sequence. To unveil the chromosomal/genomic dynamics of this multigene family in grasshoppers, we mapped U1 genes by fluorescence in situ hybridization in 70 species belonging to the families Proscopiidae, Pyrgomorphidae, Ommexechidae, Romaleidae and Acrididae. Evident clusters were observed in all species, indicating that, at least, some U1 repeats are tandemly arrayed. High conservation was observed in the first four families, with most species carrying a single U1 cluster, frequently located in the third or fourth longest autosome. By contrast, extensive variation was observed among Acrididae, from a single chromosome pair carrying U1 to all chromosome pairs carrying it, with occasional occurrence of two or more clusters in the same chromosome. DNA sequence analysis in Eyprepocnemis plorans (species carrying U1 clusters on seven different chromosome pairs) and Locusta migratoria (carrying U1 in a single chromosome pair) supported the coexistence of functional and pseudogenic lineages. One of these pseudogenic lineages was truncated in the same nucleotide position in both species, suggesting that it was present in a common ancestor to both species. At least in E. plorans, this U1 snDNA pseudogenic lineage was associated with 5S rDNA and short interspersed elements (SINE)-like mobile elements. Given that we conclude in grasshoppers that the U1 snDNA had evolved under the birth-and-death model and that its intragenomic spread might be related with mobile elements. PMID:25248465

  8. Imaging dynamic interactions between spliceosomal proteins and pre-mRNA in living cells.

    PubMed

    Rino, José; Martin, Robert M; Carvalho, Teresa; Carmo-Fonseca, Maria

    2014-02-01

    The ability to observe protein dynamics in living cells is critical for the mechanistic understanding of highly flexible biological processes such as pre-mRNA splicing by the spliceosome. Splicing relies on intricate RNA and protein networks that are repeatedly rearranged during spliceosome assembly. Here we describe a method based on fluorescence microscopy that has been used by our and other laboratories to study interaction of spliceosomal proteins with nascent pre-mRNA in living cells. The method involves co-expressing in mammalian cells the target pre-mRNA labeled with one color, and the spliceosomal protein tagged with another color. The diffusion coefficient of the protein as well as its association and dissociation rates with the pre-mRNA are estimated by fluorescence recovery after photobleaching (FRAP) or photoactivation. PMID:23969316

  9. Spliceosomal DEAH-Box ATPases Remodel Pre-mRNA to Activate Alternative Splice Sites.

    PubMed

    Semlow, Daniel R; Blanco, Mario R; Walter, Nils G; Staley, Jonathan P

    2016-02-25

    During pre-mRNA splicing, a central step in the expression and regulation of eukaryotic genes, the spliceosome selects splice sites for intron excision and exon ligation. In doing so, the spliceosome must distinguish optimal from suboptimal splice sites. At the catalytic stage of splicing, suboptimal splice sites are repressed by the DEAH-box ATPases Prp16 and Prp22. Here, using budding yeast, we show that these ATPases function further by enabling the spliceosome to search for and utilize alternative branch sites and 3' splice sites. The ATPases facilitate this search by remodeling the splicing substrate to disengage candidate splice sites. Our data support a mechanism involving 3' to 5' translocation of the ATPases along substrate RNA and toward a candidate site, but, surprisingly, not across the site. Thus, our data implicate DEAH-box ATPases in acting at a distance by pulling substrate RNA from the catalytic core of the spliceosome. PMID:26919433

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

    PubMed

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

    2015-01-01

    Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions. The network accurately captures known physical and functional associations and identifies new ones, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during spliceosome assembly. In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF, and on targets of antitumor drugs, and can be widely used to identify mechanisms of splicing regulation. PMID:25482510

  11. Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition.

    PubMed Central

    Chung, S; McLean, M R; Rymond, B C

    1999-01-01

    Mutants in the Drosophila crooked neck (crn) gene show an embryonic lethal phenotype with severe developmental defects. The unusual crn protein consists of sixteen tandem repeats of the 34 amino acid tetratricopeptide (TPR) protein recognition domain. Crn-like TPR elements are found in several RNA processing proteins, although it is unknown how the TPR repeats or the crn protein contribute to Drosophila development. We have isolated a Saccharomyces cerevisiae gene, CLF1, that encodes a crooked neck-like factor. CLF1 is an essential gene but the lethal phenotype of a clf1::HIS3 chromosomal null mutant can be rescued by plasmid-based expression of CLF1 or the Drosophila crn open reading frame. Clf1p is required in vivo and in vitro for pre-mRNA 5' splice site cleavage. Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 association. Yeast two-hybrid analyses and in vitro binding studies show that Clf1p interacts specifically and differentially with the U1 snRNP-Prp40p protein and the yeast U2AF65 homolog, Mud2p. Intriguingly, Prp40p and Mud2p also bind the phylogenetically conserved branchpoint binding protein (BBP/SF1). Our results indicate that Clf1p acts as a scaffolding protein in spliceosome assembly and suggest that Clf1p may support the cross-intron bridge during the prespliceosome-to-spliceosome transition. PMID:10445879

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

    PubMed Central

    Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C. Robertson; Xu, Xiaodong; Ma, Ligeng

    2012-01-01

    Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5′ and 3′ splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level. PMID:22942380

  13. Structural features important for the U12 snRNA binding and minor spliceosome assembly of Arabidopsis U11/U12-small nuclear ribonucleoproteins.

    PubMed

    Park, Su Jung; Jung, Hyun Ju; Nguyen Dinh, Sy; Kang, Hunseung

    2016-07-01

    Although seven proteins unique to U12 intron-specific minor spliceosomes, denoted as U11/U12-65K, -59K, -48K, -35K, -31K, -25K, and -20K, have been identified in humans and the roles of some of them have been demonstrated, the functional role of most of these proteins in plants is not understood. A recent study demonstrated that Arabidopsis U11/U12-65K is essential for U12 intron splicing and normal plant development. However, the structural features and sequence motifs important for 65 K binding to U12 snRNA and other spliceosomal proteins remain unclear. Here, we demonstrated by domain-deletion analysis that the C-terminal region of the 65 K protein bound specifically to the stem-loop III of U12 snRNA, whereas the N-terminal region of the 65 K protein was responsible for interacting with the 59 K protein. Analysis of the interactions between each snRNP protein using yeast two-hybrid analysis and in planta bimolecular fluorescence complementation and luciferase complementation imaging assays demonstrated that the core interactions among the 65 K, 59 K, and 48 K proteins were conserved between plants and animals, and multiple interactions were observed among the U11/U12-snRNP proteins. Taken together, these results reveal that U11/U12-65K is an indispensible component of the minor spliceosome complex by binding to both U11/U12-59K and U12 snRNA, and that multiple interactions among the U11/U12-snRNP proteins are necessary for minor spliceosome assembly. PMID:27232356

  14. The conserved central domain of yeast U6 snRNA: importance of U2-U6 helix Ia in spliceosome assembly.

    PubMed Central

    Ryan, Daniel E; Abelson, John

    2002-01-01

    In the pre-mRNA processing machinery of eukaryotic cells, U6 snRNA is located at or near the active site for pre-mRNA splicing catalysis, and U6 is involved in catalyzing the first chemical step of splicing. We have further defined the roles of key features of yeast U6 snRNA in the splicing process. By assaying spliceosome assembly and splicing in yeast extracts, we found that mutations of yeast U6 nt 56 and 57 are similar to previously reported deletions of U2 nt 27 or 28, all within yeast U2-U6 helix Ia. These mutations lead to the accumulation of yeast A1 spliceosomes, which form just prior to the Prp2 ATPase step and the first chemical step of splicing. These results strongly suggest that, at a late stage of spliceosome assembly, the presence of U2-U6 helix Ia is important for promoting the first chemical step of splicing, presumably by bringing together the 5' splice site region of pre-mRNA, which is base paired to U6 snRNA, and the branchsite region of the intron, which is base paired to U2 snRNA, for activation of the first chemical step of splicing, as previously proposed by Madhani and Guthrie [Cell, 1992, 71: 803-817]. In the 3' intramolecular stem-loop of U6, mutation G81C causes an allele-specific accumulation of U6 snRNP. Base pairing of the U6 3' stem-loop in yeast spliceosomes does not extend as far as to include the U6 sequence of U2-U6 helix Ib, in contrast to the human U6 3' stem-loop structure. PMID:12212854

  15. U1h shaft project

    SciTech Connect

    Brian Briggs; R. G. Musick

    2000-06-30

    The U1h shaft project is a design/build subcontract to construct one 20 foot (ft) finished diameter shaft to a depth of 1,045 ft at the Nevada Test Site. Atkinson Construction was subcontracted by Bechtel Nevada to construct the U1h Shaft for the Department of Energy. The project consists of furnishing and installing the sinking plant, construction of the 1,045 ft of concrete lined shaft, development of a shaft station at a depth of 976 ft, and construction of a loading pocket at the station. The outfitting of the shaft and installation of a new hoist may be incorporated into the project at a later date. This paper should be of interest to those involved with the construction of relatively deep shafts and underground excavations.

  16. Aberrant 3' oligoadenylation of spliceosomal U6 small nuclear RNA in poikiloderma with neutropenia.

    PubMed

    Hilcenko, Christine; Simpson, Paul J; Finch, Andrew J; Bowler, Frank R; Churcher, Mark J; Jin, Li; Packman, Len C; Shlien, Adam; Campbell, Peter; Kirwan, Michael; Dokal, Inderjeet; Warren, Alan J

    2013-02-01

    The recessive disorder poikiloderma with neutropenia (PN) is caused by mutations in the C16orf57 gene that encodes the highly conserved USB1 protein. Here, we present the 1.1 Å resolution crystal structure of human USB1, defining it as a member of the LigT-like superfamily of 2H phosphoesterases. We show that human USB1 is a distributive 3'-5' exoribonuclease that posttranscriptionally removes uridine and adenosine nucleosides from the 3' end of spliceosomal U6 small nuclear RNA (snRNA), directly catalyzing terminal 2', 3' cyclic phosphate formation. USB1 measures the appropriate length of the U6 oligo(U) tail by reading the position of a key adenine nucleotide (A102) and pausing 5 uridine residues downstream.We show that the 3' ends of U6 snRNA in PN patient lymphoblasts are elongated and unexpectedly carry nontemplated 3' oligo(A) tails that are characteristic of nuclear RNA surveillance targets. Thus, our study reveals a novel quality control pathway in which posttranscriptional 3'-end processing by USB1 protects U6 snRNA from targeting and destruction by the nuclear exosome. Our data implicate aberrant oligoadenylation of U6 snRNA in the pathogenesis of the leukemia predisposition disorder PN. PMID:23190533

  17. The Spliceosomal Phosphopeptide P140 Controls the Lupus Disease by Interacting with the HSC70 Protein and via a Mechanism Mediated by γδ T Cells

    PubMed Central

    Page, Nicolas; Schall, Nicolas; Strub, Jean-Marc; Quinternet, Marc; Chaloin, Olivier; Décossas, Marion; Cung, Manh Thong; Van Dorsselaer, Alain; Briand, Jean-Paul; Muller, Sylviane

    2009-01-01

    The phosphopeptide P140 issued from the spliceosomal U1-70K snRNP protein is recognized by lupus CD4+ T cells, transiently abolishes T cell reactivity to other spliceosomal peptides in P140-treated MRL/lpr mice, and ameliorates their clinical features. P140 modulates lupus patients' T cell response ex vivo and is currently included in phase IIb clinical trials. Its underlying mechanism of action remains elusive. Here we show that P140 peptide binds a unique cell-surface receptor, the constitutively-expressed chaperone HSC70 protein, known as a presenting-protein. P140 induces apoptosis of activated MRL/lpr CD4+ T cells. In P140-treated mice, it increases peripheral blood lymphocyte apoptosis and decreases B cell, activated T cell, and CD4−CD8−B220+ T cell counts via a specific mechanism strictly depending on γδ T cells. Expression of inflammation-linked genes is rapidly regulated in CD4+ T cells. This work led us to identify a powerful pathway taken by a newly-designed therapeutic peptide to immunomodulate lupus autoimmunity. PMID:19390596

  18. Plant Spliceosomal Introns: Not Only Cut and Paste

    PubMed Central

    Morello, L; Breviario, D

    2008-01-01

    Spliceosomal introns in higher eukaryotes are present in a high percentage of protein coding genes and represent a high proportion of transcribed nuclear DNA. In the last fifteen years, a growing mass of data concerning functional roles carried out by such intervening sequences elevated them from a selfish burden carried over by the nucleus to important active regulatory elements. Introns mediate complex gene regulation via alternative splicing; they may act in cis as expression enhancers through IME (intron-mediated enhancement of gene expression) and in trans as negative regulators through the generation of intronic microRNA. Furthermore, some introns also contain promoter sequences for alternative transcripts. Nevertheless, such regulatory roles do not require long conserved sequences, so that introns are relatively free to evolve faster than exons: this feature makes them important tools for evolutionary studies and provides the basis for the development of DNA molecular markers for polymorphisms detection. A survey of introns functions in the plant kingdom is presented. PMID:19452040

  19. Spliceosomal introns as tools for genomic and evolutionary analysis

    PubMed Central

    Irimia, Manuel; Roy, Scott William

    2008-01-01

    Over the past 5 years, the availability of dozens of whole genomic sequences from a wide variety of eukaryotic lineages has revealed a very large amount of information about the dynamics of intron loss and gain through eukaryotic history, as well as the evolution of intron sequences. Implicit in these advances is a great deal of information about the structure and evolution of surrounding sequences. Here, we review the wealth of ways in which structures of spliceosomal introns as well as their conservation and change through evolution may be harnessed for evolutionary and genomic analysis. First, we discuss uses of intron length distributions and positions in sequence assembly and annotation, and for improving alignment of homologous regions. Second, we review uses of introns in evolutionary studies, including the utility of introns as indicators of rates of sequence evolution, for inferences about molecular evolution, as signatures of orthology and paralogy, and for estimating rates of nucleotide substitution. We conclude with a discussion of phylogenetic methods utilizing intron sequences and positions. PMID:18263615

  20. A Stochastic View of Spliceosome Assembly and Recycling in the Nucleus

    PubMed Central

    Desterro, Joana M. P; Lührmann, Reinhard; Carmo-Fonseca, Maria

    2007-01-01

    How splicing factors are recruited to nascent transcripts in the nucleus in order to assemble spliceosomes on newly synthesised pre-mRNAs is unknown. To address this question, we compared the intranuclear trafficking kinetics of small nuclear ribonucleoprotein particles (snRNP) and non-snRNP proteins in the presence and absence of splicing activity. Photobleaching experiments clearly show that spliceosomal proteins move continuously throughout the entire nucleus independently of ongoing transcription or splicing. Using quantitative experimental data, a mathematical model was applied for spliceosome assembly and recycling in the nucleus. The model assumes that splicing proteins move by Brownian diffusion and interact stochastically with binding sites located at different subnuclear compartments. Inhibition of splicing, which reduces the number of pre-mRNA binding sites available for spliceosome assembly, was modeled as a decrease in the on-rate binding constant in the nucleoplasm. Simulation of microscopy experiments before and after splicing inhibition yielded results consistent with the experimental observations. Taken together, our data argue against the view that spliceosomal components are stored in nuclear speckles until a signal triggers their recruitment to nascent transcripts. Rather, the results suggest that splicing proteins are constantly diffusing throughout the entire nucleus and collide randomly and transiently with pre-mRNAs. PMID:17967051

  1. Spliceosome-mediated decay (SMD) regulates expression of nonintronic genes in budding yeast

    PubMed Central

    Volanakis, Adam; Passoni, Monica; Hector, Ralph D.; Shah, Sneha; Kilchert, Cornelia; Granneman, Sander; Vasiljeva, Lidia

    2013-01-01

    We uncovered a novel role for the spliceosome in regulating mRNA expression levels that involves splicing coupled to RNA decay, which we refer to as spliceosome-mediated decay (SMD). Our transcriptome-wide studies identified numerous transcripts that are not known to have introns but are spliced by the spliceosome at canonical splice sites in Saccharomyces cerevisiae. Products of SMD are primarily degraded by the nuclear RNA surveillance machinery. We demonstrate that SMD can significantly down-regulate mRNA levels; splicing at canonical splice sites in the bromodomain factor 2 (BDF2) transcript reduced transcript levels roughly threefold by generating unstable products that are rapidly degraded by the nuclear surveillance machinery. Regulation of BDF2 mRNA levels by SMD requires Bdf1, a functionally redundant Bdf2 paralog that plays a role in recruiting the spliceosome to the BDF2 mRNA. Interestingly, mutating BDF2 5′ splice site and branch point consensus sequences partially suppresses the bdf1Δ temperature-sensitive phenotype, suggesting that maintaining proper levels of Bdf2 via SMD is biologically important. We propose that the spliceosome can also repress protein-coding gene expression by promoting nuclear turnover of spliced RNA products and provide an insight for coordinated regulation of Bdf1 and Bdf2 levels in the cell. PMID:24065768

  2. Suppressors of a cold-sensitive mutation in yeast U4 RNA define five domains in the splicing factor Prp8 that influence spliceosome activation.

    PubMed Central

    Kuhn, A N; Brow, D A

    2000-01-01

    The highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24. PMID:10924465

  3. Use of RNA tertiary interaction modules for the crystallisation of the spliceosomal snRNP core domain.

    PubMed

    Leung, Adelaine K W; Kambach, Christian; Kondo, Yasushi; Kampmann, Martin; Jinek, Martin; Nagai, Kiyoshi

    2010-09-10

    RNA is known to perform diverse roles in the cell, often as ribonucleoprotein (RNP) particles. While the crystal structure of these RNP particles could provide crucial insights into their functions, crystallographic work on RNP complexes is often hampered by difficulties in obtaining well-diffracting crystals. The small nuclear ribonucleoprotein (snRNP) core domain, acting as an assembly nucleus for the maturation of snRNPs, plays a crucial role in the biogenesis of four of the spliceosomal snRNPs. We have succeeded in crystallising the human U4 snRNP core domain containing seven Sm proteins and a truncated U4 snRNA variant. The most critical factor in our success in the crystallisation was the introduction of various tertiary interaction modules into the RNA that could promote crystal packing without altering the core structure. Here, we describe various strategies employed in our crystallisation effort that could be applied to crystallisation of other RNP particles. PMID:20643141

  4. Mutations in U4atac snRNA, a Component of the Minor Spliceosome, in the Developmental Disorder MOPD I

    PubMed Central

    He, Huiling; Liyanarachchi, Sandya; Akagi, Keiko; Nagy, Rebecca; Li, Jingfeng; Dietrich, Rosemary C; Li, Wei; Sebastian, Nikhil; Wen, Bernard; Xin, Baozhong; Singh, Jarnail; Yan, Pearlly; Alder, Hansjuerg; Haan, Eric; Wieczorek, Dagmar; Albrecht, Beate; Puffenberger, Erik; Wang, Heng; Westman, Judith A.; Padgett, Richard A; Symer, David E; de la Chapelle, Albert

    2012-01-01

    Small nuclear RNAs (snRNAs) are essential factors in mRNA splicing. By homozygosity mapping and deep sequencing, we show that a gene encoding U4atac snRNA, a component of the minor U12-dependent spliceosome, is mutated in individuals with microcephalic osteodysplastic primordial dwarfism type I (MOPD I), a severe developmental disorder characterized by extreme intrauterine growth retardation and multiple organ abnormalities. Functional assays show that mutations (30G>A, 51G>A, 55G>A, and 111G>A) associated with MOPD I cause defective U12-dependent splicing. Endogenous U12-dependent but not U2-dependent introns are poorly spliced in MOPD I patient fibroblast cells while introduction of wild type U4atac snRNA into MOPD I cells enhances U12-dependent splicing. These results illustrate the critical role of minor intron splicing in human development. PMID:21474760

  5. Spliceosomal gene mutations in myelodysplasia: molecular links to clonal abnormalities of hematopoiesis

    PubMed Central

    Inoue, Daichi; Bradley, Robert K.; Abdel-Wahab, Omar

    2016-01-01

    Genomic analyses of the myeloid malignancies and clonal disorders of hematopoiesis that may give rise to these disorders have identified that mutations in genes encoding core spliceosomal proteins and accessory regulatory splicing factors are among the most common targets of somatic mutations. These spliceosomal mutations often occur in a mutually exclusive manner with one another and, in aggregate, account for the most frequent class of mutations in patients with myelodysplastic syndromes (MDSs) in particular. Although substantial progress has been made in understanding the effects of several of these mutations on splicing and splice site recognition, functional connections linking the mechanistic changes in splicing induced by these mutations to the phenotypic consequences of clonal and aberrant hematopoiesis are not yet well defined. This review describes our current understanding of the mechanistic and biological effects of spliceosomal gene mutations in MDSs as well as the regulation of splicing throughout normal hematopoiesis. PMID:27151974

  6. Structure of a yeast spliceosome at 3.6-angstrom resolution.

    PubMed

    Yan, Chuangye; Hang, Jing; Wan, Ruixue; Huang, Min; Wong, Catherine C L; Shi, Yigong

    2015-09-11

    Splicing of precursor messenger RNA (pre-mRNA) in yeast is executed by the spliceosome, which consists of five small nuclear ribonucleoproteins (snRNPs), NTC (nineteen complex), NTC-related proteins (NTR), and a number of associated enzymes and cofactors. Here, we report the three-dimensional structure of a Schizosaccharomyces pombe spliceosome at 3.6-angstrom resolution, revealed by means of single-particle cryogenic electron microscopy. This spliceosome contains U2 and U5 snRNPs, NTC, NTR, U6 small nuclear RNA, and an RNA intron lariat. The atomic model includes 10,574 amino acids from 37 proteins and four RNA molecules, with a combined molecular mass of approximately 1.3 megadaltons. Spp42 (Prp8 in Saccharomyces cerevisiae), the key protein component of the U5 snRNP, forms a central scaffold and anchors the catalytic center. Both the morphology and the placement of protein components appear to have evolved to facilitate the dynamic process of pre-mRNA splicing. Our near-atomic-resolution structure of a central spliceosome provides a molecular framework for mechanistic understanding of pre-mRNA splicing. PMID:26292707

  7. A protein map of the yeast activated spliceosome as obtained by electron microscopy.

    PubMed

    Sun, Chengfu; Rigo, Norbert; Fabrizio, Patrizia; Kastner, Berthold; Lührmann, Reinhard

    2016-09-01

    We have elucidated the spatial arrangement of proteins and snRNP subunits within the purified spliceosomal B(act) complex from Saccharomyces cerevisiae, using negative-stain immunoelectron microscopy. The B(act) spliceosome exhibits a mushroom-like shape with a main body connected to a foot and a steep and a shallow slope. The U5 core components, including proteins Snu114 and Prp8, are located in the main body and foot, while Brr2 is on the shallow slope. U2 snRNP components and the RNA helicase Prp2 were predominantly located in the upper regions of both slopes. While several proteins of the "nineteen complex" are located on the steep slope, Prp19, Cef1, and the U6 snRNA-binding protein Cwc2 are on the main body. Our results also indicate that the catalytic core RNP of the spliceosome resides in its main body. We thus assign distinct domains of the B(act) complex to its snRNP and protein components, and we provide first structural insights into the remodeling events at the spliceosome during its transformation from the B to the B(act) complex. PMID:27368340

  8. Spliceosome discards intermediates via the DEAH box ATPase Prp43p

    PubMed Central

    Mayas, Rabiah M.; Maita, Hiroshi; Semlow, Daniel R.; Staley, Jonathan P.

    2010-01-01

    To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal substrates that have engaged the spliceosome. Whereas DExD/H box ATPases have been implicated in rejecting suboptimal substrates, the mechanism for discarding suboptimal substrates has remained obscure. Corroborating evidence that suboptimal, mutated lariat intermediates can be exported to the cytoplasm for turnover, we have found that the ribosome can translate mutated lariat intermediates. By glycerol gradient analysis, we have found that the spliceosome can dissociate mutated lariat intermediates in vivo in a manner that requires the DEAH box ATPase Prp43p. Through an in vitro assay, we demonstrate that Prp43p promotes the discard of suboptimal and optimal 5′ exon and lariat intermediates indiscriminately. Finally, we demonstrate a requirement for Prp43p in repressing splicing at a cryptic splice site. We propose a model for the fidelity of exon ligation in which the DEAH box ATPase Prp22p slows the flow of suboptimal intermediates through exon ligation and Prp43p generally promotes discard of intermediates, thereby establishing a pathway for turnover of stalled intermediates. Because Prp43p also promotes spliceosome disassembly after exon ligation, this work establishes a parallel between the discard of suboptimal intermediates and the dissociation of a genuine excised intron product. PMID:20463285

  9. Spliceosome discards intermediates via the DEAH box ATPase Prp43p.

    PubMed

    Mayas, Rabiah M; Maita, Hiroshi; Semlow, Daniel R; Staley, Jonathan P

    2010-06-01

    To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal substrates that have engaged the spliceosome. Whereas DExD/H box ATPases have been implicated in rejecting suboptimal substrates, the mechanism for discarding suboptimal substrates has remained obscure. Corroborating evidence that suboptimal, mutated lariat intermediates can be exported to the cytoplasm for turnover, we have found that the ribosome can translate mutated lariat intermediates. By glycerol gradient analysis, we have found that the spliceosome can dissociate mutated lariat intermediates in vivo in a manner that requires the DEAH box ATPase Prp43p. Through an in vitro assay, we demonstrate that Prp43p promotes the discard of suboptimal and optimal 5' exon and lariat intermediates indiscriminately. Finally, we demonstrate a requirement for Prp43p in repressing splicing at a cryptic splice site. We propose a model for the fidelity of exon ligation in which the DEAH box ATPase Prp22p slows the flow of suboptimal intermediates through exon ligation and Prp43p generally promotes discard of intermediates, thereby establishing a pathway for turnover of stalled intermediates. Because Prp43p also promotes spliceosome disassembly after exon ligation, this work establishes a parallel between the discard of suboptimal intermediates and the dissociation of a genuine excised intron product. PMID:20463285

  10. NOUGHT MAY ENDURE BUT MUTABILITY*: SPLICEOSOME DYNAMICS AND THE REGULATION OF SPLICING

    PubMed Central

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

    2008-01-01

    SUMMARY The spliceosome is both compositionally and conformationally dynamic. Each transition along the splicing pathway presents an opportunity for progression, pausing or discard, allowing splice site choice to be regulated throughout both the assembly and catalytic phases of the reaction. PMID:18570869

  11. Single molecule analysis reveals reversible and irreversible steps during spliceosome activation

    PubMed Central

    Hoskins, Aaron A; Rodgers, Margaret L; Friedman, Larry J; Gelles, Jeff; Moore, Melissa J

    2016-01-01

    The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation. DOI: http://dx.doi.org/10.7554/eLife.14166.001 PMID:27244240

  12. The core spliceosome as target and effector of non-canonical ATM signaling

    PubMed Central

    Tresini, Maria; Warmerdam, Daniël O.; Kolovos, Petros; Snijder, Loes; Vrouwe, Mischa G.; Demmers, Jeroen A.A.; van IJcken, Wilfred F.J.; Grosveld, Frank G.; Medema, René H.; Hoeijmakers, Jan H.J.; Mullenders, Leon H.F.; Vermeulen, Wim; Marteijn, Jurgen A.

    2015-01-01

    In response to DNA damage tissue homoeostasis is ensured by protein networks promoting DNA repair, cell cycle arrest or apoptosis. DNA damage response signaling pathways coordinate these processes, partly by propagating gene expression-modulating signals. DNA damage influences not only abundance of mRNAs, but also their coding information through alternative splicing. Here we show that transcription-blocking DNA lesions promote chromatin displacement of late-stage spliceosomes and initiate a positive feedback loop centered on the signaling kinase ATM. We propose that initial spliceosome displacement and subsequent R-loop formation is triggered by pausing of RNA polymerase at DNA lesions. In turn, R-loops activate ATM which signals to further impede spliceosome organization and augment UV-triggered alternative splicing at genome-wide level. Our findings define the R-loop-dependent ATM activation by transcription-blocking lesions as an important event in the DNA damage response of non-replicating cells and highlight a key role for spliceosome displacement in this process. PMID:26106861

  13. Recruitment of the NineTeen Complex to the activated spliceosome requires AtPRMT5

    PubMed Central

    Deng, Xian; Lu, Tiancong; Wang, Lulu; Gu, Lianfeng; Sun, Jing; Kong, Xiangfeng; Liu, Chunyan; Cao, Xiaofeng

    2016-01-01

    Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is involved in a multitude of biological processes in eukaryotes. Symmetric arginine dimethylation mediated by PRMT5 modulates constitutive and alternative pre-mRNA splicing of diverse genes to regulate normal growth and development in multiple species; however, the underlying molecular mechanism remains largely unknown. A genetic screen for suppressors of an Arabidopsis symmetric arginine dimethyltransferase mutant, atprmt5, identified two gain-of-function alleles of pre-mRNA processing factor 8 gene (prp8-8 and prp8-9), the highly conserved core component of the U5 small nuclear ribonucleoprotein (snRNP) and the spliceosome. These two atprmt5 prp8 double mutants showed suppression of the developmental and splicing alterations of atprmt5 mutants. In atprmt5 mutants, the NineTeen complex failed to be assembled into the U5 snRNP to form an activated spliceosome; this phenotype was restored in the atprmt5 prp8-8 double mutants. We also found that loss of symmetric arginine dimethylation of Sm proteins prevents recruitment of the NineTeen complex and initiation of spliceosome activation. Together, our findings demonstrate that symmetric arginine dimethylation has important functions in spliceosome assembly and activation, and uncover a key molecular mechanism for arginine methylation in pre-mRNA splicing that impacts diverse developmental processes. PMID:27114555

  14. Vought O2U-1 Corsair

    NASA Technical Reports Server (NTRS)

    1930-01-01

    Vought O2U-1 Corsair: The Vought O2U-1 was the first Vought airplane to carry the name Corsair. The O2U was built as an observation aircraft for the Navy, and the example flown by the NACA for evaluation and cowling tests was one of the last O2U-1s built. This Corsair came from the Naval Reserve squadron at Naval Air Station Anacostia, Washington, D. C.

  15. U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro.

    PubMed

    Somarelli, Jason A; Mesa, Annia; Rodriguez, Carol E; Sharma, Shalini; Herrera, Rene J

    2014-04-25

    The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5' splice sites at exon/intron boundaries. U1 snRNAs associate with 5' splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the 'Smith antigen', or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs. PMID:24583175

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

    PubMed

    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

  17. Vought F4U-1 Corsair

    NASA Technical Reports Server (NTRS)

    1943-01-01

    Vought F4U-1 Corsair: This is a 'Birdcage' Corsair, so called for the canopy framing around the cockpit. Several F4Us were flown by the NACA , but this F4U-1 only flew at Langley for two months in 1943 before going to the U. S. Navy at Norfolk Naval Air Station.

  18. Vought O2U-1 Corsair

    NASA Technical Reports Server (NTRS)

    1928-01-01

    Vought O2U-1 Corsair: Suspended from the roof of the NACA's hangar at Langley Field, this Vought O2U-1 Corsair retains its float undercarriage, a contrast to other O2Us flown by the NACA which were operated on wheeled landing gear.

  19. Vought F4U-1 Corsair

    NASA Technical Reports Server (NTRS)

    1942-01-01

    Vought F4U-1 Corsair: Even though the prototype Corsair had previously been tested, this production F4U-1 also underwent trials in Langley's 30 x 60 Full Scale Tunnel. In this manner, engineers could learn what recommendations bore drag reduction 'fruit.'

  20. Vought F4U-1D Corsair

    NASA Technical Reports Server (NTRS)

    1945-01-01

    Vought F4U-1D Corsair: In February and March of 1945 this Corsair was examined in the NACA's 30 x 60 Full Scale Tunnel at Langley Field. The F4U-1D has rockets mounted on its wings for this test. After installation and during testing, the wings would be lowered to their flight position.

  1. Vought SB2U-1 Vindicator

    NASA Technical Reports Server (NTRS)

    1937-01-01

    Vought SB2U-1 Vindicator: The graceful lines of an airplane were aided by a concerted effort in drag reduction. Here a Vought SB2U-1 Vindicator is studied in Langley's 30 x 60 Full Scale Tunnel. This evaluation took place in September 1937.

  2. A small nucleolar guide RNA functions both in 2′-O-ribose methylation and pseudouridylation of the U5 spliceosomal RNA

    PubMed Central

    Jády, Beáta E.; Kiss, Tamás

    2001-01-01

    In eukaryotes, two distinct classes of small nucleolar RNAs (snoRNAs), namely the fibrillarin-associated box C/D snoRNAs and the Gar1p-associated box H/ACA snoRNAs, direct the site-specific 2′-O-ribose methylation and pseudouridylation of ribosomal RNAs (rRNAs), respectively. We have identified a novel evolutionarily conserved snoRNA, called U85, which possesses the box elements of both classes of snoRNAs and associates with both fibrillarin and Gar1p. In vitro and in vivo pseudouridylation and 2′-O-methylation experiments provide evidence that the U85 snoRNA directs 2′-O-methylation of the C45 and pseudouridylation of the U46 residues in the invariant loop 1 of the human U5 spliceosomal RNA. The U85 is the first example of a snoRNA that directs modification of an RNA polymerase II-transcribed spliceosomal RNA and that functions both in RNA pseudouridylation and 2′-O-methylation. PMID:11157760

  3. The SNW Domain of SKIP Is Required for Its Integration into the Spliceosome and Its Interaction with the Paf1 Complex in Arabidopsis.

    PubMed

    Li, Yan; Xia, Congcong; Feng, Jinlin; Yang, Dong; Wu, Fangming; Cao, Ying; Li, Legong; Ma, Ligeng

    2016-07-01

    SKIP is a conserved protein from yeasts to plants and humans. In plant cells, SKIP is a bifunctional regulator that works in the nucleus as a splicing factor by integrating into the spliceosome and as a transcriptional activator by interacting with the Paf1 complex. In this study, we identified two nuclear localization signals in SKIP and confirmed that each is sufficient to target SKIP to the nucleus. The SNW domain of SKIP is required for both its function as a splicing factor by promoting integration into the spliceosome in response to stress, and its function as a transcriptional activator by controlling its interaction with the Paf1 complex to participate in flowering. Truncated proteins that included the SNW domain and the N- or C-terminus of SKIP were still able to carry out the functions of the full-length protein in gene splicing and transcriptional activation in Arabidopsis. In addition, we found that SKIP undergoes 26S proteasome-mediated degradation, and that the C-terminus of SKIP is required to maintain the stability of the protein in plant cells. Together, our findings demonstrate the structural domain organization of SKIP and reveal the core domains and motifs underlying SKIP function in plants. PMID:27130079

  4. Network of coregulated spliceosome components revealed by zebrafish mutant in recycling factor p110

    PubMed Central

    Trede, Nikolaus S.; Medenbach, Jan; Damianov, Andrey; Hung, Lee-Hsueh; Weber, Gerhard J.; Paw, Barry H.; Zhou, Yi; Hersey, Candace; Zapata, Agustin; Keefe, Matthew; Barut, Bruce A.; Stuart, Andrew B.; Katz, Tammisty; Amemiya, Chris T.; Zon, Leonard I.; Bindereif, Albrecht

    2007-01-01

    The spliceosome cycle consists of assembly, catalysis, and recycling phases. Recycling of postspliceosomal U4 and U6 small nuclear ribonucleoproteins (snRNPs) requires p110/SART3, a general splicing factor. In this article, we report that the zebrafish earl grey (egy) mutation maps in the p110 gene and results in a phenotype characterized by thymus hypoplasia, other organ-specific defects, and death by 7 to 8 days postfertilization. U4/U6 snRNPs were disrupted in egy mutant embryos, demonstrating the importance of p110 for U4/U6 snRNP recycling in vivo. Surprisingly, expression profiling of the egy mutant revealed an extensive network of coordinately up-regulated components of the spliceosome cycle, providing a mechanism compensating for the recycling defect. Together, our data demonstrate that a mutation in a general splicing factor can lead to distinct defects in organ development and cause disease. PMID:17416673

  5. Defective minor spliceosome mRNA processing results in isolated familial growth hormone deficiency

    PubMed Central

    Argente, Jesús; Flores, Raquel; Gutiérrez-Arumí, Armand; Verma, Bhupendra; Martos-Moreno, Gabriel Á; Cuscó, Ivon; Oghabian, Ali; Chowen, Julie A; Frilander, Mikko J; Pérez-Jurado, Luis A

    2014-01-01

    The molecular basis of a significant number of cases of isolated growth hormone deficiency remains unknown. We describe three sisters affected with severe isolated growth hormone deficiency and pituitary hypoplasia caused by biallelic mutations in the RNPC3 gene, which codes for a minor spliceosome protein required for U11/U12 small nuclear ribonucleoprotein (snRNP) formation and splicing of U12-type introns. We found anomalies in U11/U12 di-snRNP formation and in splicing of multiple U12-type introns in patient cells. Defective transcripts include preprohormone convertases SPCS2 and SPCS3 and actin-related ARPC5L genes, which are candidates for the somatotroph-restricted dysfunction. The reported novel mechanism for familial growth hormone deficiency demonstrates that general mRNA processing defects of the minor spliceosome can lead to very narrow tissue-specific consequences. Subject Categories Genetics, Gene Therapy ' Genetic Disease; Metabolism PMID:24480542

  6. Phosphorylated SAP155, the spliceosomal component, is localized to chromatin in postnatal mouse testes

    SciTech Connect

    Eto, Ko; Sonoda, Yoshiyuki; Jin, Yuji; Abe, Shin-ichi

    2010-03-19

    SAP155 is an essential component of the spliceosome and its phosphorylation is required for splicing catalysis, but little is known concerning its expression and regulation during spermatogenesis in postnatal mouse testes. We report that SAP155 is ubiquitously expressed in nuclei of germ and Sertoli cells within the seminiferous tubules of 6- and 35-day postpartum (dpp) testes. Analyses by fractionation of testes revealed that (1) phosphorylated SAP155 was found in the fraction containing nuclear structures at 6 dpp in amounts much larger than that at other ages; (2) non-phosphorylated SAP155 was detected in the fraction containing nucleoplasm; and (3) phosphorylated SAP155 was preferentially associated with chromatin. Our findings suggest that the active spliceosome, containing phosphorylated SAP155, performs pre-mRNA splicing on chromatin concomitant with transcription during testicular development.

  7. Origin of a peculiar extra U(1)

    SciTech Connect

    Barr, S.M.; Dorsner, I.

    2005-07-01

    The origin of a family-independent ''extra U(1)'', discovered by Barr, Bednarz, and Benesh and independently by Ma, and whose phenomenology has recently been studied by Ma and Roy, is discussed. Even though it satisfies anomaly constraints in a highly economical way, with just a single extra triplet of leptons per family, this extra U(1) cannot come from four-dimensional grand unification. However, it is shown here that it can come from a Pati-Salam scheme with an extra U(1), which explains the otherwise surprising cancellation of anomalies.

  8. Structure of a yeast catalytic step I spliceosome at 3.4 Å resolution.

    PubMed

    Wan, Ruixue; Yan, Chuangye; Bai, Rui; Huang, Gaoxingyu; Shi, Yigong

    2016-08-26

    Each cycle of pre-messenger RNA splicing, carried out by the spliceosome, comprises two sequential transesterification reactions, which result in the removal of an intron and the joining of two exons. Here we report an atomic structure of a catalytic step I spliceosome (known as the C complex) from Saccharomyces cerevisiae, as determined by cryo-electron microscopy at an average resolution of 3.4 angstroms. In the structure, the 2'-OH of the invariant adenine nucleotide in the branch point sequence (BPS) is covalently joined to the phosphate at the 5' end of the 5' splice site (5'SS), forming an intron lariat. The freed 5' exon remains anchored to loop I of U5 small nuclear RNA (snRNA), and the 5'SS and BPS of the intron form duplexes with conserved U6 and U2 snRNA sequences, respectively. Specific placement of these RNA elements at the catalytic cavity of Prp8 is stabilized by 15 protein components, including Snu114 and the splicing factors Cwc21, Cwc22, Cwc25, and Yju2. These features, representing the conformation of the spliceosome after the first-step reaction, predict structural changes that are needed for the execution of the second-step transesterification reaction. PMID:27445308

  9. The exosome controls alternative splicing by mediating the gene expression and assembly of the spliceosome complex

    PubMed Central

    Zhang, Lin; Wan, Yufeng; Huang, Guobin; Wang, Dongni; Yu, Xinyang; Huang, Guocun; Guo, Jinhu

    2015-01-01

    The exosome is a complex with exoribonuclease activity that regulates RNA surveillance and turnover. The exosome also plays a role in regulating the degradation of precursor mRNAs to maintain the expression of splicing variants. In Neurospora, the silencing of rrp44, which encodes the catalytic subunit of the exosome, changed the expression of a set of spliceosomal snRNA, snRNP genes and SR protein related genes. The knockdown of rrp44 also affected the assembly of the spliceosome. RNA-seq analysis revealed a global change in bulk splicing events. Exosome-mediated splicing may regulate alternative splicing of NCU05290, NCU07421 and the circadian clock gene frequency (frq). The knockdown of rrp44 led to an increased ratio of splicing variants without intron 6 (I-6) and shorter protein isoform small FRQ (s-FRQ) as a consequence. These findings suggest that the exosome controls splicing events by regulating the degradation of precursor mRNAs and the gene expression, assembly and function of the spliceosome. PMID:26306464

  10. Vought SB2U-1 Vindicator

    NASA Technical Reports Server (NTRS)

    1938-01-01

    Vought SB2U-1 Vindicator: This Vought SB2U-1 Vindicator was acquired for one month in late 1938 from NAS Anacostia, Washington, D. C. Anacostia was the source of many of the naval aircraft flown by the NACA, in part due to its proximity and in part became it was the Navy's flight test base. The nose slot cowling was aimed at improving engine cooling.

  11. Vought XF7U-1 Cutlass

    NASA Technical Reports Server (NTRS)

    1948-01-01

    Vought XF7U-1 Cutlass: This is the first Vought Cutlass, the XF7U-1. This tailless fighter, powered by two Westinghouse J34 turbojets, arrived at Langley in December 1948. Aircraft number NAVY 122472. Wing detail, servicing, fueling, nose detail, front view, side view, side view, side view with ladder, 3/4 rear view, rear view, interior of cockpit, nose strake, exterior of cockpit with pilot, pilot climbing out of aircraft.

  12. The importin-β binding domain of snurportin1 is responsible for the Ran- and energy-independent nuclear import of spliceosomal U snRNPs in vitro

    PubMed Central

    Huber, Jochen; Dickmanns, Achim; Lührmann, Reinhard

    2002-01-01

    The nuclear localization signal (NLS) of spliceosomal U snRNPs is composed of the U snRNA's 2,2,7-trimethyl-guanosine (m3G)-cap and the Sm core domain. The m3G-cap is specifically bound by snurportin1, which contains an NH2-terminal importin-β binding (IBB) domain and a COOH-terminal m3G-cap–binding region that bears no structural similarity to known import adaptors like importin-α (impα). Here, we show that recombinant snurportin1 and importin-β (impβ) are not only necessary, but also sufficient for U1 snRNP transport to the nuclei of digitonin-permeabilized HeLa cells. In contrast to impα–dependent import, single rounds of U1 snRNP import, mediated by the nuclear import receptor complex snurportin1–impβ, did not require Ran and energy. The same Ran- and energy-independent import was even observed for U5 snRNP, which has a molecular weight of more than one million. Interestingly, in the presence of impβ and a snurportin1 mutant containing an impα IBB domain (IBBimpα), nuclear U1 snRNP import was Ran dependent. Furthermore, β-galactosidase (βGal) containing a snurportin1 IBB domain, but not IBBimpα-βGal, was imported into the nucleus in a Ran-independent manner. Our results suggest that the nature of the IBB domain modulates the strength and/or site of interaction of impβ with nucleoporins of the nuclear pore complex, and thus whether or not Ran is required to dissociate these interactions. PMID:11815630

  13. Multiple components of the spliceosome regulate Mcl1 activity in neuroblastoma

    PubMed Central

    Laetsch, T W; Liu, X; Vu, A; Sliozberg, M; Vido, M; Elci, O U; Goldsmith, K C; Hogarty, M D

    2014-01-01

    Cancer treatments induce cell stress to trigger apoptosis in tumor cells. Many cancers repress these apoptotic signals through alterations in the Bcl2 proteins that regulate this process. Therapeutics that target these specific survival biases are in development, and drugs that inhibit Bcl2 activities have shown clinical activity for some cancers. Mcl1 is a survival factor for which no effective antagonists have been developed, so it remains a principal mediator of therapy resistance, including to Bcl2 inhibitors. We used a synthetic-lethal screening strategy to identify genes that regulate Mcl1 survival activity using the pediatric tumor neuroblastoma (NB) as a model, as a large subset are functionally verified to be Mcl1 dependent and Bcl2 inhibitor resistant. A targeted siRNA screen identified genes whose knockdown restores sensitivity of Mcl1-dependent NBs to ABT-737, a small molecule inhibitor of Bcl2, BclXL and BclW. Three target genes that shifted the ABT-737 IC50 >1 log were identified and validated: PSMD14, UBL5 and PRPF8. The latter two are members of a recently characterized subcomplex of the spliceosome that along with SART1 is responsible for non-canonical 5′-splice sequence recognition in yeast. We showed that SART1 knockdown similarly sensitized Mcl1-dependent NB to ABT-737 and that triple knockdown of UBL5/PRPF8/SART1 phenocopied direct MCL1 knockdown, whereas having no effect on Bcl2-dependent NBs. Both genetic spliceosome knockdown or treatment with SF3b-interacting spliceosome inhibitors like spliceostatin A led to preferential pro-apoptotic Mcl1-S splicing and reduced translation and abundance of Mcl1 protein. In contrast, BN82865, which inhibits the second transesterification step in terminal spliceosome processing, did not have this effect. These findings demonstrate a prominent role for the spliceosome in mediating Mcl1 activity and suggest that drugs that target either the specific UBL5/PRPF8/SART1 subcomplex or SF3b functions may have a

  14. The spliceosome assembly factor GEMIN2 attenuates the effects of temperature on alternative splicing and circadian rhythms

    PubMed Central

    Schlaen, Rubén Gustavo; Mancini, Estefanía; Sanchez, Sabrina Elena; Perez-Santángelo, Soledad; Rugnone, Matías L.; Simpson, Craig G.; Brown, John W. S.; Zhang, Xu; Chernomoretz, Ariel; Yanovsky, Marcelo J.

    2015-01-01

    The mechanisms by which poikilothermic organisms ensure that biological processes are robust to temperature changes are largely unknown. Temperature compensation, the ability of circadian rhythms to maintain a relatively constant period over the broad range of temperatures resulting from seasonal fluctuations in environmental conditions, is a defining property of circadian networks. Temperature affects the alternative splicing (AS) of several clock genes in fungi, plants, and flies, but the splicing factors that modulate these effects to ensure clock accuracy throughout the year remain to be identified. Here we show that GEMIN2, a spliceosomal small nuclear ribonucleoprotein assembly factor conserved from yeast to humans, modulates low temperature effects on a large subset of pre-mRNA splicing events. In particular, GEMIN2 controls the AS of several clock genes and attenuates the effects of temperature on the circadian period in Arabidopsis thaliana. We conclude that GEMIN2 is a key component of a posttranscriptional regulatory mechanism that ensures the appropriate acclimation of plants to daily and seasonal changes in temperature conditions. PMID:26170331

  15. Chemical modifications of ribonuclease U1.

    PubMed

    Hashimoto, J; Takahashi, K

    1977-04-01

    In order to obtain information on the nature of the amino acid residues involved in the activity of ribonuclease U1 [EC 3.1.4.8], various chemical modifications of the enzyme were carried out. RNase U1 was inactivated by reaction with iodoacetate at pH 5.5 with concomitant incorporation of 1 carboxymethyl group per molecule of the enzyme. The residue specifically modified by iodoacetate was identified as one of the glutamic acid residues, as in the case of RNase T1. The enzyme was also inactivated extensively by reaction with iodoacetamide at pH 8.0 with the loss of about one residue each of histidine and lysine. When RNase U1 was treated with a large excess of phenylglyoxal, the enzymatic activity and binding ability toward 3'-GMP were lost, with simultaneous modification of about 1 residue of arginine. The reaction of citraconic anhydride with RNase U1 led to the loss of enzymatic activity and modification of about 1 residue of lysine. The inactivated enzyme, however, retained binding ability toward 3'-GMP. These results indicate that there are marked similarities in the active sites of RNases T1 and U1. PMID:18450

  16. Vought O3U-1 Corsair

    NASA Technical Reports Server (NTRS)

    1931-01-01

    Vought O3U-1 Corsair: This aircraft, the Vought O3U-1 Corsair, was the first aircraft tested in the Full Scale Tunnel. It is shown here during preliminary tests in the FST before the balance was enclosed. NACA engineers checked the lift and drag characteristics of several aircraft with the results of earlier flight tests. Smith DeFrance concluded NACA TR No. 459, 'The agreement that has been obtained between the flight and full-scale tunnel results, together with the consistent manner in which measurements can be repeated when check tests are made, has demonstrated the accuracy and value of the equipment for aeronautical research.' (p. 298)

  17. Meta-analysis of gene expression profiles indicates genes in spliceosome pathway are up-regulated in hepatocellular carcinoma (HCC).

    PubMed

    Xu, Weijin; Huang, Huixing; Yu, Long; Cao, Lihuan

    2015-04-01

    Hepatocellular carcinoma (HCC) is among the commonest kind of malignant tumors, which accounts for more than 500,000 cases of newly diagnosed cancer annually. Many microarray studies for identifying differentially expressed genes (DEGs) in HCC have been conducted, but results have varied across different studies. Here, we performed a meta-analysis of publicly available microarray Gene Expression Omnibus datasets, which covers five independent studies, containing 753 HCC samples and 638 non-tumor liver samples. We identified 192 DEGs that were consistently up-regulated in HCC vs. normal liver tissue. For the 192 up-regulated genes, we performed Kyoto Encyclopedia of Genes and Genomes pathway analysis. To our surprise, besides several cell growth-related pathways, spliceosome pathway was also up-regulated in HCC. For further exploring the relationship between spliceosome pathway and HCC, we investigated the expression data of spliceosome pathway genes in 15 independent studies in Nextbio database ( https://www.nextbio.com/b/nextbioCorp.nb ). It was found that many genes of spliceosome pathway such as HSPA1A, SNRPE, SF3B2, SF3B4 and TRA2A genes which we identified to be up-regulated in our meta-analysis were generally overexpressed in HCC. At last, using real-time PCR, we also found that BUD31, SF3B2, SF3B4, SNRPE, SPINK1, TPA2A and HSPA1A genes are significantly up-regulated in clinical HCC samples when compared to the corresponding non-tumorous liver tissues. Our study for the first time indicates that many genes of spliceosome pathway are up-regulated in HCC. This finding might put new insights for people's understanding about the relationship of spliceosome pathway and HCC. PMID:25731616

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

    PubMed Central

    Crous, Pedro W.; de Wit, Pierre J. G. M.; van der Burgt, Ate

    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 identified novel ILEs in eight additional fungal species that are phylogenetically related to F. fulva and D. septosporum using PCR amplification with primers derived from previously identified ILEs. The ILE content appeared unique to each species, suggesting independent multiplication events. Interestingly, we identified four genes each containing two gained ILEs. By analysing intron positions in orthologues of these four genes in Ascomycota, we found that three ILEs had inserted within a 15 bp window that contains regular spliceosomal introns in other fungal species. These three positions are not the result of intron sliding because ILEs are newly gained introns. Furthermore, the alternative hypothesis of an inferred ancestral gain followed by independent losses contradicts the observed degeneration of ILEs. These observations clearly indicate three parallel intron gains in four genes that were randomly identified. Our findings suggest that parallel intron gain is a phenomenon that has been highly underestimated in ILE-containing fungi, and likely in the whole fungal kingdom. PMID:26046656

  19. Crystal structures of a group II intron maturase reveal a missing link in spliceosome evolution.

    PubMed

    Zhao, Chen; Pyle, Anna Marie

    2016-06-01

    Group II introns are self-splicing ribozymes that are essential in many organisms, and they have been hypothesized to share a common evolutionary ancestor with the spliceosome. Although structural similarity of RNA components supports this connection, it is of interest to determine whether associated protein factors also share an evolutionary heritage. Here we present the crystal structures of reverse transcriptase (RT) domains from two group II intron-encoded proteins (maturases) from Roseburia intestinalis and Eubacterium rectale, obtained at 1.2-Å and 2.1-Å resolution, respectively. These domains are more similar in architecture to the spliceosomal Prp8 RT-like domain than to any other RTs, and they share substantial similarity with flaviviral RNA polymerases. The RT domain itself is sufficient for binding intron RNA with high affinity and specificity, and it is contained within an active RT enzyme. These studies provide a foundation for understanding structure-function relationships within group II intron-maturase complexes. PMID:27136328

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

    PubMed

    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

  1. Structure of a yeast activated spliceosome at 3.5 Å resolution.

    PubMed

    Yan, Chuangye; Wan, Ruixue; Bai, Rui; Huang, Gaoxingyu; Shi, Yigong

    2016-08-26

    Pre-messenger RNA (pre-mRNA) splicing is carried out by the spliceosome, which undergoes an intricate assembly and activation process. Here, we report an atomic structure of an activated spliceosome (known as the B(act) complex) from Saccharomyces cerevisiae, determined by cryo-electron microscopy at an average resolution of 3.52 angstroms. The final refined model contains U2 and U5 small nuclear ribonucleoprotein particles (snRNPs), U6 small nuclear RNA (snRNA), nineteen complex (NTC), NTC-related (NTR) protein, and a 71-nucleotide pre-mRNA molecule, which amount to 13,505 amino acids from 38 proteins and a combined molecular mass of about 1.6 megadaltons. The 5' exon is anchored by loop I of U5 snRNA, whereas the 5' splice site (5'SS) and the branch-point sequence (BPS) of the intron are specifically recognized by U6 and U2 snRNA, respectively. Except for coordination of the catalytic metal ions, the RNA elements at the catalytic cavity of Prp8 are mostly primed for catalysis. The catalytic latency is maintained by the SF3b complex, which encircles the BPS, and the splicing factors Cwc24 and Prp11, which shield the 5' exon-5'SS junction. This structure, together with those determined earlier, outlines a molecular framework for the pre-mRNA splicing reaction. PMID:27445306

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

    PubMed Central

    Shah, Priya; Pozzi, Berta; Gebhard, Leopoldo G.; Mammi, Pablo; Yanovsky, Marcelo J.; Andino, Raul; Krogan, Nevan; Srebrow, Anabella; Gamarnik, Andrea V.

    2016-01-01

    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

  3. Spliceosome SNRNP200 Promotes Viral RNA Sensing and IRF3 Activation of Antiviral Response

    PubMed Central

    Tremblay, Nicolas; Baril, Martin; Chatel-Chaix, Laurent; Es-Saad, Salwa; Park, Alex Young; Koenekoop, Robert K.; Lamarre, Daniel

    2016-01-01

    Spliceosomal SNRNP200 is a Ski2-like RNA helicase that is associated with retinitis pigmentosa 33 (RP33). Here we found that SNRNP200 promotes viral RNA sensing and IRF3 activation through the ability of its amino-terminal Sec63 domain (Sec63-1) to bind RNA and to interact with TBK1. We show that SNRNP200 relocalizes into TBK1-containing cytoplasmic structures upon infection, in contrast to the RP33-associated S1087L mutant, which is also unable to rescue antiviral response of SNRNP200 knockdown cells. This functional rescue correlates with the Sec63-1-mediated binding of viral RNA. The hindered IFN-β production of knockdown cells was further confirmed in peripheral blood cells of RP33 patients bearing missense mutation in SNRNP200 upon infection with Sendai virus (SeV). This work identifies a novel immunoregulatory role of the spliceosomal SNRNP200 helicase as an RNA sensor and TBK1 adaptor for the activation of IRF3-mediated antiviral innate response. PMID:27454487

  4. Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing.

    PubMed

    Trochet, Delphine; Prudhon, Bernard; Jollet, Arnaud; Lorain, Stéphanie; Bitoun, Marc

    2016-01-01

    Dynamin 2 (DNM2) is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3'-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5'-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development. PMID:27623444

  5. Spliceosome SNRNP200 Promotes Viral RNA Sensing and IRF3 Activation of Antiviral Response.

    PubMed

    Tremblay, Nicolas; Baril, Martin; Chatel-Chaix, Laurent; Es-Saad, Salwa; Park, Alex Young; Koenekoop, Robert K; Lamarre, Daniel

    2016-07-01

    Spliceosomal SNRNP200 is a Ski2-like RNA helicase that is associated with retinitis pigmentosa 33 (RP33). Here we found that SNRNP200 promotes viral RNA sensing and IRF3 activation through the ability of its amino-terminal Sec63 domain (Sec63-1) to bind RNA and to interact with TBK1. We show that SNRNP200 relocalizes into TBK1-containing cytoplasmic structures upon infection, in contrast to the RP33-associated S1087L mutant, which is also unable to rescue antiviral response of SNRNP200 knockdown cells. This functional rescue correlates with the Sec63-1-mediated binding of viral RNA. The hindered IFN-β production of knockdown cells was further confirmed in peripheral blood cells of RP33 patients bearing missense mutation in SNRNP200 upon infection with Sendai virus (SeV). This work identifies a novel immunoregulatory role of the spliceosomal SNRNP200 helicase as an RNA sensor and TBK1 adaptor for the activation of IRF3-mediated antiviral innate response. PMID:27454487

  6. Supergravity solutions without triholomorphic U(1) isometries

    SciTech Connect

    Ghezelbash, A. M.

    2008-12-15

    We investigate the construction of five-dimensional supergravity solutions that do not have any triholomorphic U(1) isometries. We construct a class of solutions that in various limits of parameters reduces to many of previously constructed five-dimensional supergravity solutions based on both hyper-Kaehler base spaces that can be put into a Gibbons-Hawking form and hyper-Kaehler base spaces that cannot be put into a Gibbons-Hawking form. We find a new solution which is over triaxial Bianchi type IX Einstein-hyper-Kaehler base space with no triholomorphic U(1) symmetry. One special case of this solution corresponds to a five-dimensional solution based on Eguchi-Hanson type II geometry.

  7. More modular invariant anomalous U(1) breaking

    SciTech Connect

    Gaillard, Mary K.; Giedt, Joel

    2002-06-27

    We consider the case of several scalar fields, charged under a number of U(1) factors, acquiring vacuum expectation values due to anomalous U(1). We demonstrate how to make redefinitions at the superfield level in order to account for tree-level exchange of vector supermultiplets in the effective supergravity theory of the light fields in the supersymmetric vacuum phase. Our approach builds up on previous results that we obtained in a more elementary case. We find that the modular weights of light fields are typically shifted from their original values, allowing an interpretation in terms of the preservation of modular invariance in the effective theory. We address various subtleties in defining unitary gauge that are associated with the noncanonical Kahler potential of modular invariant supergravity, the vacuum degeneracy, and the role of the dilaton field. We discuss the effective superpotential for the light fields and note how proton decay operators may be obtained when the heavy fields are integrated out of the theory at the tree-level. We also address how our formalism may be extended to describe the generalized Green-Schwarz mechanism for multiple anomalous U(1)'s that occur in four-dimensional Type I and Type IIB string constructions.

  8. Retrofitted natural supersymmetry from a U(1)

    NASA Astrophysics Data System (ADS)

    Hardy, Edward; March-Russell, John

    2013-05-01

    We propose that a single, spontaneously broken, U(1) gauge symmetry may be responsible for suppressing both the first two generation Yukawa couplings, and also, in a correlated manner, parameters in the dynamical supersymmetry (SUSY) breaking sector by the mechanism of retrofitting. In the dynamical SUSY breaking sector, these small parameters are typically required in order to introduce R-symmetry breaking in a controlled manner and obtain phenomenologically viable meta-stable vacua. The heavy U(1) multiplet mediates a dominant contribution to the first two generation MSSM sfermion soft masses, while gauge mediation provides a parametrically suppressed soft term contribution to the stop and most other states, so realising a natural SUSY spectrum in a fashion consistent with SUSY unification. In explicit models the spectra obtained can be such that current LHC limits are evaded, and predictions of flavour changing processes are consistent with observation. We examine both implementations with low scale mediation, and string-motivated examples where the U(1) is anomalous before the inclusion of a generalised Green-Schwarz mechanism.

  9. Tetramers reveal IL-17-secreting CD4+ T cells that are specific for U1-70 in lupus and mixed connective tissue disease.

    PubMed

    Kattah, Nicole H; Newell, Evan W; Jarrell, Justin Ansel; Chu, Alvina D; Xie, Jianming; Kattah, Michael G; Goldberger, Ofir; Ye, Jessica; Chakravarty, Eliza F; Davis, Mark M; Utz, Paul J

    2015-03-10

    Antigen-specific CD4(+) T cells are implicated in the autoimmune disease systemic lupus erythematosus (SLE), but little is known about the peptide antigens that they recognize and their precise function in disease. We generated a series of MHC class II tetramers of I-E(k)-containing peptides from the spliceosomal protein U1-70 that specifically stain distinct CD4(+) T-cell populations in MRL/lpr mice. The T-cell populations recognize an epitope differing only by the presence or absence of a single phosphate residue at position serine(140). The frequency of CD4(+) T cells specific for U1-70(131-150):I-E(k) (without phosphorylation) correlates with disease severity and anti-U1-70 autoantibody production. These T cells also express RORγt and produce IL-17A. Furthermore, the U1-70-specific CD4(+) T cells that produce IL-17A are detected in a subset of patients with SLE and are significantly increased in patients with mixed connective tissue disease. These studies provide tools for studying antigen-specific CD4(+) T cells in lupus, and demonstrate an antigen-specific source of IL-17A in autoimmune disease. PMID:25713364

  10. The network organization of protein interactions in the spliceosome is reproduced by the simple rules of food-web models.

    PubMed

    Pires, Mathias M; Cantor, Maurício; Guimarães, Paulo R; de Aguiar, Marcus A M; Dos Reis, Sérgio F; Coltri, Patricia P

    2015-01-01

    The network structure of biological systems provides information on the underlying processes shaping their organization and dynamics. Here we examined the structure of the network depicting protein interactions within the spliceosome, the macromolecular complex responsible for splicing in eukaryotic cells. We show the interactions of less connected spliceosome proteins are nested subsets of the connections of the highly connected proteins. At the same time, the network has a modular structure with groups of proteins sharing similar interaction patterns. We then investigated the role of affinity and specificity in shaping the spliceosome network by adapting a probabilistic model originally designed to reproduce food webs. This food-web model was as successful in reproducing the structure of protein interactions as it is in reproducing interactions among species. The good performance of the model suggests affinity and specificity, partially determined by protein size and the timing of association to the complex, may be determining network structure. Moreover, because network models allow building ensembles of realistic networks while encompassing uncertainty they can be useful to examine the dynamics and vulnerability of intracelullar processes. Unraveling the mechanisms organizing the spliceosome interactions is important to characterize the role of individual proteins on splicing catalysis and regulation. PMID:26443080

  11. The network organization of protein interactions in the spliceosome is reproduced by the simple rules of food-web models

    PubMed Central

    Pires, Mathias M.; Cantor, Maurício; Guimarães, Paulo R.; de Aguiar, Marcus A. M.; dos Reis, Sérgio F.; Coltri, Patricia P.

    2015-01-01

    The network structure of biological systems provides information on the underlying processes shaping their organization and dynamics. Here we examined the structure of the network depicting protein interactions within the spliceosome, the macromolecular complex responsible for splicing in eukaryotic cells. We show the interactions of less connected spliceosome proteins are nested subsets of the connections of the highly connected proteins. At the same time, the network has a modular structure with groups of proteins sharing similar interaction patterns. We then investigated the role of affinity and specificity in shaping the spliceosome network by adapting a probabilistic model originally designed to reproduce food webs. This food-web model was as successful in reproducing the structure of protein interactions as it is in reproducing interactions among species. The good performance of the model suggests affinity and specificity, partially determined by protein size and the timing of association to the complex, may be determining network structure. Moreover, because network models allow building ensembles of realistic networks while encompassing uncertainty they can be useful to examine the dynamics and vulnerability of intracelullar processes. Unraveling the mechanisms organizing the spliceosome interactions is important to characterize the role of individual proteins on splicing catalysis and regulation. PMID:26443080

  12. Confined vortices in topologically massive U (1U (1 ) theory

    NASA Astrophysics Data System (ADS)

    Anber, Mohamed M.; Burnier, Yannis; Sabancilar, Eray; Shaposhnikov, Mikhail

    2015-09-01

    We report on a new topological vortex solution in U (1U (1 ) Maxwell-Chern-Simons theory. The existence of the vortex is envisaged by analytical means, and a numerical solution is obtained by integrating the equations of motion. These vortices have a long-range force because one of the U(1)'s remains unbroken in the infrared, which is guarded by the Coleman-Hill theorem. The sum of the winding numbers of an ensemble of vortices has to vanish; otherwise the system would have a logarithmically divergent energy. In turn, these vortices exhibit classical confinement. We investigate the rich parameter space of the solutions, and show that one recovers the Abrikosov-Nielsen-Olesen, U(1) Maxwell-Chern-Simons, U(1) pure Chern-Simons, and global vortices as various limiting cases. Unlike these limiting cases, the higher winding solutions of our vortices carry noninteger charges under the broken U(1). This is the first vortex solution exhibiting such behavior.

  13. Vought O3U-1 'Corsair'

    NASA Technical Reports Server (NTRS)

    1931-01-01

    Vought O3U-1 'Corsair' in Full-Scale Tunnel (FST). This photograph was taken in September 1931 after the balance had been enclosed. This aircraft was also used earlier during the summer for preliminary tests in the FST and as the subject of some of the first publicity photographs taken of FST operations. NACA engineers checked the lift and drag characteristics of several aircraft with the results of earlier flight tests. Smith DeFrance concluded NACA TR No. 459, 'The agreement that has been obtained between the flight and full-scale tunnel results, together with the consistent manner in which measurements can be repeated when check tests are made, has demonstrated the accuracy and value of the equipment for aeronautical research.' (p. 298)

  14. Anomalous Flavor U(1)_X for Everything

    SciTech Connect

    Dreiner, Herbi K.; Murayama, Hitoshi; Thormeier, Marc

    2003-12-01

    We present an ambitious model of flavor, based on an anomalous U(1)_X gauge symmetry with one flavon, only two right-handed neutrinos and only two mass scales: M_{grav} and m_{3/2}. In particular, there are no new scales introduced for right-handed neutrino masses. The X-charges of the matter fields are such that R-parity is conserved exactly, higher-dimensional operators are sufficiently suppressed to guarantee a proton lifetime in agreement with experiment, and the phenomenology is viable for quarks, charged leptons, as well as neutrinos. In our model one of the three light neutrinos automatically is massless. The price we have to pay for this very successful model are highly fractional X-charges which can likely be improved with less restrictive phenomenological ansatze for mass matrices.

  15. U(1) problem at finite temperature

    SciTech Connect

    Janik, Romuald A.; Nowak, Maciej A.; Papp, Gabor; Zahed, Ismail

    1999-11-22

    We model the effects of a large number of zero and near-zero modes in the QCD partition function by using sparse chiral matrix models with an emphasis on the quenched topological susceptibility in the choice of the measure. At finite temperature, the zero modes are not affected by temperature but are allowed to pair into topologically neutral near-zero modes which are gapped at high temperature. In equilibrium, chiral and U(1) symmetry are simultaneously restored for total pairing, evading mean-field arguments. We analyze a number of susceptibilities versus the light quark masses. At the transition point the topological susceptibility vanishes, and the dependence on the vacuum angle {theta} drops out. Our results are briefly contrasted with recent lattice simulations.

  16. Anomalies, U(1)' and the MSSM

    NASA Astrophysics Data System (ADS)

    Racioppi, Antonio

    2009-07-01

    This Thesis reviews an extension of the MSSM by the addition of an anomalous abelian vector multiplet and contains some original results concerning the phenomenology of an anomalous Z'. The review part covers an introduction of the MSSM focusing on its main features, a discussion on the chiral anomalies and how to cancel them in the Standard Model and by the Green-Schwarz mechanism. Then, the original results are presented. We build the Lagrangian for the Minimal Anomalous U(1)' Extension of the MSSM where the anomalies are cancelled by the Green-Schwarz mechanism and the addition of Chern-Simons terms, stressing the main differences between our model and the MSSM. The advantage of this choice over the standard one is that it allows for arbitrary values of the quantum numbers of the extra U(1). As a first step towards the study of hadron annihilations producing four leptons in the final state (a clean signal which might be studied at LHC) we then compute the decays Z'to Z_0 g and Z'to Z_0 Z_0. We find that the largest values of the decay rate are sim 10^{-4} GeV, while the expected number of events per year at LHC is at most of the order of 10. Then we compute the relic density predicted by our model with a new dark matter candidate, the axino, which is the LSP of the theory. We find agreement with experimental data admitting a bino-higgsino NLSP or a wino-like NLSP, almost degenerate in mass to the axino.

  17. The target of the DEAH-box NTP triphosphatase Prp43 in Saccharomyces cerevisiae spliceosomes is the U2 snRNP-intron interaction

    PubMed Central

    Fourmann, Jean-Baptiste; Dybkov, Olexandr; Agafonov, Dmitry E; Tauchert, Marcel J; Urlaub, Henning; Ficner, Ralf; Fabrizio, Patrizia; Lührmann, Reinhard

    2016-01-01

    The DEAH-box NTPase Prp43 and its cofactors Ntr1 and Ntr2 form the NTR complex and are required for disassembling intron-lariat spliceosomes (ILS) and defective earlier spliceosomes. However, the Prp43 binding site in the spliceosome and its target(s) are unknown. We show that Prp43 fused to Ntr1's G-patch motif (Prp43_Ntr1GP) is as efficient as the NTR in ILS disassembly, yielding identical dissociation products and recognizing its natural ILS target even in the absence of Ntr1’s C-terminal-domain (CTD) and Ntr2. Unlike the NTR, Prp43_Ntr1GP disassembles earlier spliceosomal complexes (A, B, Bact), indicating that Ntr2/Ntr1-CTD prevents NTR from disrupting properly assembled spliceosomes other than the ILS. The U2 snRNP-intron interaction is disrupted in all complexes by Prp43_Ntr1GP, and in the spliceosome contacts U2 proteins and the pre-mRNA, indicating that the U2 snRNP-intron interaction is Prp43’s major target. DOI: http://dx.doi.org/10.7554/eLife.15564.001 PMID:27115347

  18. Calabi-Yau manifolds from noncommutative Hermitian U (1 ) instantons

    NASA Astrophysics Data System (ADS)

    Yang, Hyun Seok

    2015-05-01

    We show that Calabi-Yau manifolds are emergent from the commutative limit of six-dimensional noncommutative Hermitian U (1 ) instantons. Therefore, we argue that the noncommutative Hermitian U (1 ) instantons correspond to quantized Calabi-Yau manifolds.

  19. Substrate-assisted mechanism of RNP disruption by the spliceosomal Brr2 RNA helicase.

    PubMed

    Theuser, Matthias; Höbartner, Claudia; Wahl, Markus C; Santos, Karine F

    2016-07-12

    The Brr2 RNA helicase disrupts the U4/U6 di-small nuclear RNA-protein complex (di-snRNP) during spliceosome activation via ATP-driven translocation on the U4 snRNA strand. However, it is unclear how bound proteins influence U4/U6 unwinding, which regions of the U4/U6 duplex the helicase actively unwinds, and whether U4/U6 components are released as individual molecules or as subcomplexes. Here, we set up a recombinant Brr2-mediated U4/U6 di-snRNP disruption system, showing that sequential addition of the U4/U6 proteins small nuclear ribonucleoprotein-associated protein 1 (Snu13), pre-mRNA processing factor 31 (Prp31), and Prp3 to U4/U6 di-snRNA leads to a stepwise decrease of Brr2-mediated U4/U6 unwinding, but that unwinding is largely restored by a Brr2 cofactor, the C-terminal Jab1/MPN domain of the Prp8 protein. Brr2-mediated U4/U6 unwinding was strongly inhibited by mutations in U4/U6 di-snRNAs that diminish the ability of U6 snRNA to adopt an alternative conformation but leave the number and kind of U4/U6 base pairs unchanged. Irrespective of the presence of the cofactor, the helicase segregated a Prp3-Prp31-Snu13-U4/U6 RNP into an intact Prp31-Snu13-U4 snRNA particle, free Prp3, and free U6 snRNA. Together, these observations suggest that Brr2 translocates only a limited distance on the U4 snRNA strand and does not actively release RNA-bound proteins. Unwinding is then completed by the partially displaced U6 snRNA adopting an alternative conformation, which leads to dismantling of the Prp3-binding site on U4/U6 di-snRNA but leaves the Prp31- and Snu13-binding sites on U4 snRNA unaffected. In this fashion, Brr2 can activate the spliceosome by stripping U6 snRNA of all precatalytic binding partners, while minimizing logistic requirements for U4/U6 di-snRNP reassembly after splicing. PMID:27354531

  20. Evidence for a group II intron-like catalytic triplex in the spliceosome

    PubMed Central

    Piccirilli, Joseph A.; Staley, Jonathan P.

    2014-01-01

    To catalyze pre-mRNA splicing, U6 snRNA positions two metals that interact directly with the scissile phosphates. The U6 metal ligands correspond stereospecifically to metal ligands within the catalytic domain V of a group II self-splicing intron. In domain V, the ligands are organized by base-triple interactions, which also juxtapose the 3′ splice site with the catalytic metals. However, in the spliceosome, the mechanism for organizing catalytic metals and recruiting the substrate has remained unclear. Here we show by genetics, crosslinking, and biochemistry in yeast that analogous triples form in U6 and promote catalytic metal binding and both chemical steps of splicing. Because the triples include an element that defines the 5′ splice site, the triples also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals. Our data indicate that U6 adopts a group II intron-like tertiary conformation to catalyze splicing. PMID:24747940

  1. Proteomic Analysis Reveals CACN-1 Is a Component of the Spliceosome in Caenorhabditis elegans

    PubMed Central

    Doherty, Michael F.; Adelmant, Guillaume; Cecchetelli, Alyssa D.; Marto, Jarrod A.; Cram, Erin J.

    2014-01-01

    Cell migration is essential for embryonic development and tissue formation in all animals. cacn-1 is a conserved gene of unknown molecular function identified in a genome-wide screen for genes that regulate distal tip cell migration in the nematode worm Caenorhabditis elegans. In this study we take a proteomics approach to understand CACN-1 function. To isolate CACN-1−interacting proteins, we used an in vivo tandem-affinity purification strategy. Tandem-affinity purification−tagged CACN-1 complexes were isolated from C. elegans lysate, analyzed by mass spectrometry, and characterized bioinformatically. Results suggest significant interaction of CACN-1 with the C. elegans spliceosome. All of the identified interactors were screened for distal tip cell migration phenotypes using RNAi. Depletion of many of these factors led to distal tip cell migration defects, particularly a failure to stop migrating, a phenotype commonly seen in cacn-1 deficient animals. The results of this screen identify eight novel regulators of cell migration and suggest CACN-1 may participate in a protein network dedicated to high-fidelity gonad development. The composition of proteins comprising the CACN-1 network suggests that this critical developmental module may exert its influence through alternative splicing or other post-transcriptional gene regulation. PMID:24948787

  2. Impact of allogeneic hematopoietic cell transplant in patients with myeloid neoplasms carrying spliceosomal mutations.

    PubMed

    Hamilton, Betty Ky; Visconte, Valeria; Jia, Xuefei; Tabarroki, Ali; Makishima, Hideki; Hasrouni, Edy; Abounader, Donna; Kalaycio, Matt; Sekeres, Mikkael A; Sobecks, Ronald; Duong Liu, Hien; Bolwell, Brian; Maciejewski, Jaroslaw P; Copelan, Edward; Tiu, Ramon V

    2016-06-01

    Molecular predictors of outcome are increasingly important in determining optimal therapy for myeloid neoplasms. Mutations in the spliceosomal genes (U2AF1 and SRSF2) predict for poor outcomes in myelodysplastic syndromes (MDS) and related diseases. We investigated the effect of hematopoietic cell transplant (HCT) on the negative prognostic impact of U2AF1 and SRSF2 mutations. In total, 122 patients with MDS (30%), acute myeloid leukemia (51%), myeloproliferative neoplasms (MPN) (11%), and MDS/MPN (8%) receiving a HCT from 2003 to 2012 were evaluated for mutations in U2AF1 and SRSF2 by direct sequencing. Median time of follow up was 24 months (range 0.46-110). SRSF2 mutations were detected in 11 (10%) patients and U2AF1 in 3 (3%) patients. There were no significant differences in baseline characteristics between mutated and wild-type (WT) patients. Patients carrying SRSF2 and U2AF1 mutations had similar overall survival (P = 0.84), relapse mortality (P = 0.50), and non-relapse mortality (P = 0.72) compared to WT patients. However, taking into account disease status and cytogenetics in a subset of AML patients, SRSF2 and U2AF1 mutations were associated with worse survival (HR 3.71, P = 0.035). Am. J. Hematol. 91:406-409, 2016. © 2016 Wiley Periodicals, Inc. PMID:26799334

  3. Repair of Rhodopsin mRNA by Spliceosome-Mediated RNA Trans-Splicing: A New Approach for Autosomal Dominant Retinitis Pigmentosa

    PubMed Central

    Berger, Adeline; Lorain, Stéphanie; Joséphine, Charlène; Desrosiers, Melissa; Peccate, Cécile; Voit, Thomas; Garcia, Luis; Sahel, José-Alain; Bemelmans, Alexis-Pierre

    2015-01-01

    The promising clinical results obtained for ocular gene therapy in recent years have paved the way for gene supplementation to treat recessively inherited forms of retinal degeneration. The situation is more complex for dominant mutations, as the toxic mutant gene product must be removed. We used spliceosome-mediated RNA trans-splicing as a strategy for repairing the transcript of the rhodopsin gene, the gene most frequently mutated in autosomal dominant retinitis pigmentosa. We tested 17 different molecules targeting the pre-mRNA intron 1, by transient transfection of HEK-293T cells, with subsequent trans-splicing quantification at the transcript level. We found that the targeting of some parts of the intron promoted trans-splicing more efficiently than the targeting of other areas, and that trans-splicing rate could be increased by modifying the replacement sequence. We then developed cell lines stably expressing the rhodopsin gene, for the assessment of phenotypic criteria relevant to the pathogenesis of retinitis pigmentosa. Using this model, we showed that trans-splicing restored the correct localization of the protein to the plasma membrane. Finally, we tested our best candidate by AAV gene transfer in a mouse model of retinitis pigmentosa that expresses a mutant allele of the human rhodopsin gene, and demonstrated the feasibility of trans-splicing in vivo. This work paves the way for trans-splicing gene therapy to treat retinitis pigmentosa due to rhodopsin gene mutation and, more generally, for the treatment of genetic diseases with dominant transmission. PMID:25619725

  4. Repair of rhodopsin mRNA by spliceosome-mediated RNA trans-splicing: a new approach for autosomal dominant retinitis pigmentosa.

    PubMed

    Berger, Adeline; Lorain, Stéphanie; Joséphine, Charlène; Desrosiers, Melissa; Peccate, Cécile; Voit, Thomas; Garcia, Luis; Sahel, José-Alain; Bemelmans, Alexis-Pierre

    2015-05-01

    The promising clinical results obtained for ocular gene therapy in recent years have paved the way for gene supplementation to treat recessively inherited forms of retinal degeneration. The situation is more complex for dominant mutations, as the toxic mutant gene product must be removed. We used spliceosome-mediated RNA trans-splicing as a strategy for repairing the transcript of the rhodopsin gene, the gene most frequently mutated in autosomal dominant retinitis pigmentosa. We tested 17 different molecules targeting the pre-mRNA intron 1, by transient transfection of HEK-293T cells, with subsequent trans-splicing quantification at the transcript level. We found that the targeting of some parts of the intron promoted trans-splicing more efficiently than the targeting of other areas, and that trans-splicing rate could be increased by modifying the replacement sequence. We then developed cell lines stably expressing the rhodopsin gene, for the assessment of phenotypic criteria relevant to the pathogenesis of retinitis pigmentosa. Using this model, we showed that trans-splicing restored the correct localization of the protein to the plasma membrane. Finally, we tested our best candidate by AAV gene transfer in a mouse model of retinitis pigmentosa that expresses a mutant allele of the human rhodopsin gene, and demonstrated the feasibility of trans-splicing in vivo. This work paves the way for trans-splicing gene therapy to treat retinitis pigmentosa due to rhodopsin gene mutation and, more generally, for the treatment of genetic diseases with dominant transmission. PMID:25619725

  5. Structural Basis of Brr2-Prp8 Interactions and Implications for U5 snRNP Biogenesis and the Spliceosome Active Site

    PubMed Central

    Nguyen, Thi Hoang Duong; Li, Jade; Galej, Wojciech P.; Oshikane, Hiroyuki; Newman, Andrew J.; Nagai, Kiyoshi

    2013-01-01

    Summary The U5 small nuclear ribonucleoprotein particle (snRNP) helicase Brr2 disrupts the U4/U6 small nuclear RNA (snRNA) duplex and allows U6 snRNA to engage in an intricate RNA network at the active center of the spliceosome. Here, we present the structure of yeast Brr2 in complex with the Jab1/MPN domain of Prp8, which stimulates Brr2 activity. Contrary to previous reports, our crystal structure and mutagenesis data show that the Jab1/MPN domain binds exclusively to the N-terminal helicase cassette. The residues in the Jab1/MPN domain, whose mutations in human Prp8 cause the degenerative eye disease retinitis pigmentosa, are found at or near the interface with Brr2, clarifying its molecular pathology. In the cytoplasm, Prp8 forms a precursor complex with U5 snRNA, seven Sm proteins, Snu114, and Aar2, but after nuclear import, Brr2 replaces Aar2 to form mature U5 snRNP. Our structure explains why Aar2 and Brr2 are mutually exclusive and provides important insights into the assembly of U5 snRNP. PMID:23727230

  6. The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.

    PubMed

    Absmeier, Eva; Wollenhaupt, Jan; Mozaffari-Jovin, Sina; Becke, Christian; Lee, Chung-Tien; Preussner, Marco; Heyd, Florian; Urlaub, Henning; Lührmann, Reinhard; Santos, Karine F; Wahl, Markus C

    2015-12-15

    The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation. PMID:26637280

  7. The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

    PubMed Central

    Absmeier, Eva; Wollenhaupt, Jan; Mozaffari-Jovin, Sina; Becke, Christian; Lee, Chung-Tien; Preussner, Marco; Heyd, Florian; Urlaub, Henning; Lührmann, Reinhard; Santos, Karine F.; Wahl, Markus C.

    2015-01-01

    The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation. PMID:26637280

  8. The MUC1 Extracellular Domain Subunit Is Found in Nuclear Speckles and Associates with Spliceosomes

    PubMed Central

    Kumar, Priyadarsina; Ji, Jennifer W.; Martsching, Lindsay; Douglas, Gordon C.

    2012-01-01

    MUC1 is a large transmembrane glycoprotein and oncogene expressed by epithelial cells and overexpressed and underglycosylated in cancer cells. The MUC1 cytoplasmic subunit (MUC1-C) can translocate to the nucleus and regulate gene expression. It is frequently assumed that the MUC1 extracellular subunit (MUC1-N) does not enter the nucleus. Based on an unexpected observation that MUC1 extracellular domain antibody produced an apparently nucleus-associated staining pattern in trophoblasts, we have tested the hypothesis that MUC1-N is expressed inside the nucleus. Three different antibodies were used to identify MUC1-N in normal epithelial cells and tissues as well as in several cancer cell lines. The results of immunofluorescence and confocal microscopy analyses as well as subcellular fractionation, Western blotting, and siRNA/shRNA studies, confirm that MUC1-N is found within nuclei of all cell types examined. More detailed examination of its intranuclear distribution using a proximity ligation assay, subcellular fractionation, and immunoprecipitation suggests that MUC1-N is located in nuclear speckles (interchromatin granule clusters) and closely associates with the spliceosome protein U2AF65. Nuclear localization of MUC1-N was abolished when cells were treated with RNase A and nuclear localization was altered when cells were incubated with the transcription inhibitor 5,6-dichloro-1-b-d-ribofuranosylbenzimidazole (DRB). While MUC1-N predominantly associated with speckles, MUC1-C was present in the nuclear matrix, nucleoli, and the nuclear periphery. In some nuclei, confocal microscopic analysis suggest that MUC1-C staining is located close to, but only partially overlaps, MUC1-N in speckles. However, only MUC1-N was found in isolated speckles by Western blotting. Also, MUC1-C and MUC1-N distributed differently during mitosis. These results suggest that MUC1-N translocates to the nucleus where it is expressed in nuclear speckles and that MUC1-N and MUC1-C have

  9. Molecular dynamics studies of U1A-RNA complexes.

    PubMed Central

    Reyes, C M; Kollman, P A

    1999-01-01

    The U1A protein binds to a hairpin RNA and an internal-loop RNA with picomolar affinities. To probe the molecular basis of U1A binding, we performed state-of-the-art nanosecond molecular dynamics simulations on both complexes. The good agreement with experimental structures supports the protocols used in the simulations. We compare the dynamics, hydrogen-bonding occupancies, and interfacial flexibility of both complexes and also describe a rigid-body motion in the U1A-internal loop complex that is not observed in the U1A-hairpin simulation. We relate these observations to experimental mutational studies and highlight their significance in U1A binding affinity and specificity. PMID:10024175

  10. Mapping epitopes of U1-70K autoantibodies at single-amino acid resolution.

    PubMed

    Haddon, David James; Jarrell, Justin Ansel; Diep, Vivian K; Wand, Hannah E; Price, Jordan V; Tangsombatvisit, Stephanie; Credo, Grace M; Mackey, Sally; Dekker, Cornelia L; Baechler, Emily C; Liu, Chih Long; Varma, Madoo; Utz, Paul J

    2015-01-01

    The mechanisms underlying development of ribonucleoprotein (RNP) autoantibodies are unclear. The U1-70K protein is the predominant target of RNP autoantibodies, and the RNA binding domain has been shown to be the immunodominant autoantigenic region of U1-70K, although the specific epitopes are not known. To precisely map U1-70K epitopes, we developed silicon-based peptide microarrays with >5700 features, corresponding to 843 unique peptides derived from the U1-70K protein. The microarrays feature overlapping peptides, with single-amino acid resolution in length and location, spanning amino acids 110-170 within the U1-70K RNA binding domain. We evaluated the serum IgG of a cohort of patients with systemic lupus erythematosus (SLE; n = 26) using the microarrays, and identified multiple reactive epitopes, including peptides 116-121 and 143-148. Indirect peptide ELISA analysis of the sera of patients with SLE (n = 88) revealed that ∼14% of patients had serum IgG reactivity to 116-121, while reactivity to 143-148 appeared to be limited to a single patient. SLE patients with serum reactivity to 116-121 had significantly lower SLE Disease Activity Index (SLEDAI) scores at the time of sampling, compared to non-reactive patients. Minimal reactivity to the peptides was observed in the sera of healthy controls (n = 92). Competitive ELISA showed antibodies to 116-121 bind a common epitope in U1-70K (68-72) and the matrix protein M1 of human influenza B viruses. Institutional Review Boards approved this study. Knowledge of the precise epitopes of U1-70K autoantibodies may provide insight into the mechanisms of development of anti-RNP, identify potential clinical biomarkers and inform ongoing clinical trails of peptide-based therapeutics. PMID:26333287

  11. Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

    PubMed Central

    Bartlett, J S; Sethna, M; Ramamurthy, L; Gowen, S A; Samulski, R J; Marzluff, W F

    1996-01-01

    Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8799116

  12. Gauge U(1) dark symmetry and radiative light fermion masses

    NASA Astrophysics Data System (ADS)

    Kownacki, Corey; Ma, Ernest

    2016-09-01

    A gauge U (1) family symmetry is proposed, spanning the quarks and leptons as well as particles of the dark sector. The breaking of U (1) to Z2 divides the two sectors and generates one-loop radiative masses for the first two families of quarks and leptons, as well as all three neutrinos. We study the phenomenological implications of this new connection between family symmetry and dark matter. In particular, a scalar or pseudoscalar particle associated with this U (1) breaking may be identified with the 750 GeV diphoton resonance recently observed at the Large Hadron Collider (LHC).

  13. Structural Basis of the Recruitment of Ubiquitin-specific Protease USP15 by Spliceosome Recycling Factor SART3.

    PubMed

    Zhang, Qi; Harding, Rachel; Hou, Feng; Dong, Aiping; Walker, John R; Bteich, Joseph; Tong, Yufeng

    2016-08-12

    Ubiquitin-specific proteases (USPs) USP15 and USP4 belong to a subset of USPs featuring an N-terminal tandem domain in USP (DUSP) and ubiquitin-like (UBL) domain. Squamous cell carcinoma antigen recognized by T-cell 3 (SART3), a spliceosome recycling factor, binds to the DUSP-UBL domain of USP15 and USP4, recruiting them to the nucleus from the cytosol to control deubiquitination of histone H2B and spliceosomal proteins, respectively. To provide structural insight, we solved crystal structures of SART3 in the apo-form and in complex with the DUSP-UBL domain of USP15 at 2.0 and 3.0 Å, respectively. Structural analysis reveals SART3 contains 12 half-a-tetratricopeptide (HAT) repeats, organized into two subdomains, HAT-N and HAT-C. SART3 dimerizes through the concave surface of HAT-C, whereas the HAT-C convex surface binds USP15 in a novel bipartite mode. Isothermal titration calorimetry measurements and mutagenesis analysis confirmed key residues of USP15 involved in the interaction and indicated USP15 binds 20-fold stronger than USP4. PMID:27255711

  14. RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

    PubMed Central

    Chang, T H; Clark, M W; Lustig, A J; Cusick, M E; Abelson, J

    1988-01-01

    The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus. Images PMID:3043176

  15. Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

    NASA Technical Reports Server (NTRS)

    Reddy, A. S.; Czernik, A. J.; An, G.; Poovaiah, B. W.

    1992-01-01

    We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

  16. Simple U (1 ) gauge theory explanation of the diphoton excess

    NASA Astrophysics Data System (ADS)

    Chang, Spencer

    2016-03-01

    The recent ATLAS and CMS diphoton resonance excesses are explored in a simple U (1 ) gauge theory extension of the Standard Model where the resonance is the Higgs boson of the U (1 ) symmetry breaking, ϕ . This particle couples to exotic quarks which, through loops, can produce a large enough rate to explain the excess. Due to the choice of U (1 ) charges, flavor constraints are naturally suppressed, allowing arbitrary flavor violation in the decays of the new quarks to up-type quarks, modifying their signal topologies. An additional heavy quark in the model decays to the lighter exotic quark by emitting either ϕ or the U (1 ) gauge boson Ax, giving extra signals containing diphoton and digluon resonances. Finally, the new Higgs can decay into γ Ax and Z Ax, followed by Ax decaying into Standard Model fermions through kinetic mixing. Thus, this model gives interesting modified signals to the general class of exotic quark models explaining the diphoton resonance.

  17. Underground storage tank 291-D1U1: Closure plan

    SciTech Connect

    Mancieri, S.; Giuntoli, N.

    1993-09-01

    The 291-D1U1 tank system was installed in 1983 on the north side of Building 291. It supplies diesel fuel to the Building 291 emergency generator and air compressor. The emergency generator and air compressor are located southwest and southeast, respectively, of the tank (see Appendix B, Figure 2). The tank system consists of a single-walled, 2,000- gallon, fiberglass tank and a fuel pump system, fill pipe, vent pipe, electrical conduit, and fuel supply and return piping. The area to be excavated is paved with asphalt and concrete. It is not known whether a concrete anchor pad is associated with this tank. Additionally, this closure plan assumes that the diesel tank is below the fill pad. The emergency generator and air compressor for Building 291 and its associated UST, 291-D1U1, are currently in use. The generator and air compressor will be supplied by a temporary above-ground fuel tank prior to the removal of 291-D1U1. An above-ground fuel tank will be installed as a permanent replacement for 291-D1U1. The system was registered with the State Water Resources Control Board on June 27, 1984, as 291-41D and has subsequently been renamed 291-D1U1. Figure 1 (see Appendix B) shows the location of the 291-D1U1 tank system in relation to the Lawrence Livermore National Laboratory (LLNL). Figure 2 (see Appendix B) shows the 291-D1U1 tank system in relation to Building 291. Figure 3 (see Appendix B) shows a plan view of the 291-D1U1 tank system.

  18. Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.

    PubMed

    Philippi, Susanne; Lorain, Stéphanie; Beley, Cyriaque; Peccate, Cécile; Précigout, Guillaume; Spuler, Simone; Garcia, Luis

    2015-07-15

    The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dystrophy 1 (MMD1). We established a 3' replacement strategy for mutated DYSF pre-mRNAs induced by spliceosome-mediated pre-mRNA trans-splicing (SmaRT) by the use of a PTM. In contrast to previously established SmaRT strategies, we particularly focused on the identification of a suitable pre-mRNA target intron other than the optimization of the PTM design. By targeting DYSF pre-mRNA introns harbouring differentially defined 3' splice sites (3' SS), we found that target introns encoding weakly defined 3' SSs were trans-spliced successfully in vitro in human LGMD2B myoblasts as well as in vivo in skeletal muscle of wild-type and Dysf(-/-) mice. For the first time, we demonstrate rescue of Dysf protein by SmaRT in vivo. Moreover, we identified concordant qualities among the successfully targeted Dysf introns and targeted endogenous introns in previously reported SmaRT approaches that might facilitate a selective choice of target introns in future SmaRT strategies. PMID:25904108

  19. Meta-Analysis and Gene Set Analysis of Archived Microarrays Suggest Implication of the Spliceosome in Metastatic and Hypoxic Phenotypes

    PubMed Central

    Bareke, Eric; Depiereux, Sophie; Michiels, Carine; Depiereux, Eric

    2014-01-01

    We propose to make use of the wealth of underused DNA chip data available in public repositories to study the molecular mechanisms behind the adaptation of cancer cells to hypoxic conditions leading to the metastatic phenotype. We have developed new bioinformatics tools and adapted others to identify with maximum sensitivity those genes which are expressed differentially across several experiments. The comparison of two analytical approaches, based on either Over Representation Analysis or Functional Class Scoring, by a meta-analysis-based approach, led to the retrieval of known information about the biological situation – thus validating the model – but also more importantly to the discovery of the previously unknown implication of the spliceosome, the cellular machinery responsible for mRNA splicing, in the development of metastasis. PMID:24497970

  20. The evolution of single-copy Drosophila nuclear 4f-rnp genes: spliceosomal intron losses create polymorphic alleles.

    PubMed

    Feiber, Amy L; Rangarajan, Janaki; Vaughn, Jack C

    2002-10-01

    This study provides the first report in which spliceosomal intron losses within a single-copy gene create functional polymorphic alleles in a population. 4f-rnp has previously been shown to be a nuclear gene that is localized on the X chromosome in D. melanogaster and to have eight short spliceosomal introns. An insect species survey was done via polymerase chain reaction (PCR) amplification and sequencing of a 1028-bp gene fragment spanning introns 4-8, which are located in the 3' half of the gene. The results show that 4f-rnp and (thus far) introns 7 and 8 are at least as old as order Odonata (dragonflies), an early-diverging insect line. Unexpectedly, several species within the dipteran family Drosophilidae were found to contain two differently sized 4f-rnp gene sequence variants, owing to precise in-frame intron losses. Results of single-male D. melanogaster PCR analyses show that the two gene size variants are allelic and that the intron loss mechanism appears to be biased toward the 3' end of the gene. A stable potential stem-loop has been identified in D. melanogaster, predicted to fold the 4f-rnp mRNA 3' terminus into a natural primer for subsequent reverse transcription into cDNA. When results are displayed in a phylogenetic context, multiple independent intron loss events are identified. These observations support a model in which frequently occurring cDNAs have led to numerous independent intron losses via homologous recombination/gene conversion during 4f-rnp gene evolution. The results provide insights into the evolution of intron loss and may lead to improved understanding of the dynamics of this process in natural populations. PMID:12355261

  1. On the Partical Conservation of the U(1) Current

    NASA Astrophysics Data System (ADS)

    Kawarabayashi, K.; Ohta, N.

    1981-11-01

    Recently proposed partial conservation of the U(1) current(PCU1C) is applied to estimate the decay rates of various OZIforbidden processes. The results obtained are in good agreement withexperiments and thus indicate the important role played by the U(1)axial-vector anomaly in these decay processes. Octet Jp = (1/2)+ baryons are next introduced into this scheme and low energy theorems related to the θ dependence of the matrix elements are investigated. Physical consequences of non-zero θ (strong CP-violation) are also discussed with the help of the PCU1C. The results are used to give the bound on θ.

  2. Inflationary magnetogenesis with broken local U(1) symmetry

    NASA Astrophysics Data System (ADS)

    Domènech, Guillem; Lin, Chunshan; Sasaki, Misao

    2016-07-01

    We point out that a successful inflationary magnetogenesis could be realised if we break the local U(1) gauge symmetry during inflation. The effective electric charge is fixed as a fundamental constant, which allows us to obtain an almost scale-invariant magnetic spectrum avoiding both the strong coupling and back reaction problems. We examine the corrections to the primordial curvature perturbation due to these stochastic electromagnetic fields and find that, at both linear and non-linear orders, the contributions from the electromagnetic field are negligible compared to those created from vacuum fluctuations. Finally, the U(1) gauge symmetry is restored at the end of inflation.

  3. In vitro synthesis of vertebrate U1 snRNA.

    PubMed Central

    Lund, E; Dahlberg, J E

    1989-01-01

    We have developed a DNA-dependent in vitro transcription system for vertebrate snRNA genes. By isolating the nuclei (germinal vesicles, GVs) of Xenopus laevis oocytes under oil to maintain the in vivo composition of their internal milieu, we are able to prepare nuclei that retain their ability to synthesize snRNAs efficiently. Homogenates of these GVs synthesize correctly initiated and terminated U1 snRNA using exogenous X.laevis U1 genes as templates. The templates may be either injected into the nucleus prior to its isolation or added to the nuclear homogenate. Images PMID:2714253

  4. Time-Reversal Symmetric U (1 ) Quantum Spin Liquids

    NASA Astrophysics Data System (ADS)

    Wang, Chong; Senthil, T.

    2016-01-01

    We study possible quantum U (1 ) spin liquids in three dimensions with time-reversal symmetry. We find a total of seven families of such U (1 ) spin liquids, distinguished by the properties of their emergent electric or magnetic charges. We show how these spin liquids are related to each other. Two of these classes admit nontrivial protected surface states which we describe. We show how to access all of the seven spin liquids through slave particle (parton) constructions. We also provide intuitive loop gas descriptions of their ground-state wave functions. One of these phases is the "topological Mott insulator," conventionally described as a topological insulator of an emergent fermionic "spinon." We show that this phase admits a remarkable dual description as a topological insulator of emergent fermionic magnetic monopoles. This results in a new (possibly natural) surface phase for the topological Mott insulator and a new slave particle construction. We describe some of the continuous quantum phase transitions between the different U (1 ) spin liquids. Each of these seven families of states admits a finer distinction in terms of their surface properties, which we determine by combining these spin liquids with symmetry-protected topological phases. We discuss lessons for materials such as pyrochlore quantum spin ices which may harbor a U (1 ) spin liquid. We suggest the topological Mott insulator as a possible ground state in some range of parameters for the quantum spin ice Hamiltonian.

  5. Progressive gauge U(1) family symmetry for quarks and leptons

    NASA Astrophysics Data System (ADS)

    Ma, Ernest

    2016-08-01

    The pattern of quark and lepton mass matrices is unexplained in the standard model of particle interactions. I propose the novel idea of a progressive gauge U (1 ) symmetry where it is a reflection of the regressive electroweak symmetry breaking pattern, caused by an extended Higgs scalar sector. Phenomenological implications of this new hypothesis are discussed.

  6. U(1) gauge symmetry breaking in a charged closed universe

    SciTech Connect

    Kim, J.E. ); Lee, T. )

    1990-10-30

    In this paper, the authors obtain the consistency condition on a U(1) gauge boson mass in a charged closed universe, m{sup 2} = 8{pi}GJ{sup 0}J{sub 0}/(R {minus} 2{Lambda}), where J{sup 0} is the charge density.

  7. Underground storage tank 511-D1U1 closure plan

    SciTech Connect

    Mancieri, S.; Giuntoli, N.

    1993-09-01

    This document contains the closure plan for diesel fuel underground storage tank 511-D1U1 and appendices containing supplemental information such as staff training certification and task summaries. Precision tank test data, a site health and safety plan, and material safety data sheets are also included.

  8. H2B Ubiquitylation Modulates Spliceosome Assembly and Function in Budding Yeast

    PubMed Central

    Hérissant, Lucas; Moehle, Erica A.; Bertaccini, Diego; Van Dorsselaer, Alain; Schaeffer-Reiss, Christine; Guthrie, Christine; Dargemont, Catherine

    2014-01-01

    Background information Commitment to splicing occurs co-transcriptionally, but a major unanswered question is the extent to which various modifications of chromatin, the template for transcription in vivo, contribute to the regulation of splicing. Results Here we perform genome-wide analyses showing that inhibition of specific marks – H2B ubiquitylation, H3K4 methylation, and H3K36 methylation – perturbs splicing in budding yeast, with each modification exerting gene-specific effects. Furthermore, semi-quantitative mass spectrometry on purified nuclear mRNPs and chromatin immunoprecipitation analysis on intron-containing genes indicated that H2B ubiquitylation, but not Set1-, Set2- or Dot1-dependent H3 methylation, stimulates recruitment of the early splicing factors, namely U1 and U2 snRNPs, onto nascent RNAs. Conclusions These results suggest that histone modifications impact splicing of distinct subsets of genes using distinct pathways. PMID:24476359

  9. Spliceosomal gene mutations are frequent events in the diverse mutational spectrum of chronic myelomonocytic leukemia but largely absent in juvenile myelomonocytic leukemia

    PubMed Central

    Kar, Sarah Abu; Jankowska, Anna; Makishima, Hideki; Visconte, Valeria; Jerez, Andres; Sugimoto, Yuka; Muramatsu, Hideki; Traina, Fabiola; Afable, Manuel; Guinta, Kathryn; Tiu, Ramon V.; Przychodzen, Bartlomiej; Sakaguchi, Hirotoshi; Kojima, Seiji; Sekeres, Mikkael A.; List, Alan F.; McDevitt, Michael A.; Maciejewski, Jaroslaw P.

    2013-01-01

    Chronic myelomonocytic leukemia is a heterogeneous disease with multifactorial molecular pathogenesis. Various recurrent somatic mutations have been detected alone or in combination in chronic myelomonocytic leukemia. Recently, recurrent mutations in spliceosomal genes have been discovered. We investigated the contribution of U2AF1, SRSF2 and SF3B1 mutations in the pathogenesis of chronic myelomonocytic leukemia and closely related diseases. We genotyped a cohort of patients with chronic myelomonocytic leukemia, secondary acute myeloid leukemia derived from chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia for somatic mutations in U2AF1, SRSF2, SF3B1 and in the other 12 most frequently affected genes in these conditions. Chromosomal abnormalities were assessed by nucleotide polymorphism array-based karyotyping. The presence of molecular lesions was correlated with clinical endpoints. Mutations in SRSF2, U2AF1 and SF3B1 were found in 32%, 13% and 6% of cases of chronic myelomonocytic leukemia, secondary acute myeloid leukemia derived from chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia, respectively. Spliceosomal genes were affected in various combinations with other mutations, including TET2, ASXL1, CBL, EZH2, RAS, IDH1/2, DNMT3A, TP53, UTX and RUNX1. Worse overall survival was associated with mutations in U2AF1 (P=0.047) and DNMT3A (P=0.015). RAS mutations had an impact on overall survival in secondary acute myeloid leukemia (P=0.0456). By comparison, our screening of juvenile myelomonocytic leukemia cases showed mutations in ASXL1 (4%), CBL (10%), and RAS (6%) but not in IDH1/2, TET2, EZH2, DNMT3A or the three spliceosomal genes. SRSF2 and U2AF1 along with TET2 (48%) and ASXL1 (38%) are frequently affected by somatic mutations in chronic myelomonocytic leukemia, quite distinctly from the profile seen in juvenile myelomonocytic leukemia. Our data also suggest that spliceosomal mutations are of ancestral origin. PMID:22773603

  10. The 3.8 Å structure of the U4/U6.U5 tri-snRNP: Insights into spliceosome assembly and catalysis.

    PubMed

    Wan, Ruixue; Yan, Chuangye; Bai, Rui; Wang, Lin; Huang, Min; Wong, Catherine C L; Shi, Yigong

    2016-01-29

    Splicing of precursor messenger RNA is accomplished by a dynamic megacomplex known as the spliceosome. Assembly of a functional spliceosome requires a preassembled U4/U6.U5 tri-snRNP complex, which comprises the U5 small nuclear ribonucleoprotein (snRNP), the U4 and U6 small nuclear RNA (snRNA) duplex, and a number of protein factors. Here we report the three-dimensional structure of a Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at an overall resolution of 3.8 angstroms by single-particle electron cryomicroscopy. The local resolution for the core regions of the tri-snRNP reaches 3.0 to 3.5 angstroms, allowing construction of a refined atomic model. Our structure contains U5 snRNA, the extensively base-paired U4/U6 snRNA, and 30 proteins including Prp8 and Snu114, which amount to 8495 amino acids and 263 nucleotides with a combined molecular mass of ~1 megadalton. The catalytic nucleotide U80 from U6 snRNA exists in an inactive conformation, stabilized by its base-pairing interactions with U4 snRNA and protected by Prp3. Pre-messenger RNA is bound in the tri-snRNP through base-pairing interactions with U6 snRNA and loop I of U5 snRNA. This structure, together with that of the spliceosome, reveals the molecular choreography of the snRNAs in the activation process of the spliceosomal ribozyme. PMID:26743623

  11. Regular spliceosomal introns are invasive in Chlamydomonas reinhardtii: 15 introns in the recently relocated mitochondrial cox2 and cox3 genes.

    PubMed

    Watanabe, K I; Ohama, T

    2001-01-01

    In the unicellular green alga, Chlamydomonas reinhardtii, cytochrome oxidase subunit 2 (cox2) and 3 (cox3) genes are missing from the mitochondrial genome. We isolated and sequenced a BAC clone that carries the whole cox3 gene and its corresponding cDNA. Almost the entire cox2 gene and its cDNA were also determined. Comparison of the genomic and the corresponding cDNA sequences revealed that the cox3 gene contains as many as nine spliceosomal introns and that cox2 bears six introns. Putative mitochondria targeting signals were predicted at each N terminal of the cox genes. These spliceosomal introns were typical GT-AG-type introns, which are very common not only in Chlamydomonas nuclear genes but also in diverse eukaryotic taxa. We found no particular distinguishing features in the cox introns. Comparative analysis of these genes with the various mitochondrial genes showed that 8 of the 15 introns were interrupting the conserved mature protein coding segments, while the other 7 introns were located in the N-terminal target peptide regions. Phylogenetic analysis of the evolutionary position of C. reinhardtii in Chlorophyta was carried out and the existence of the cox2 and cox3 genes in the mitochondrial genome was superimposed in the tree. This analysis clearly shows that these cox genes were relocated during the evolution of Chlorophyceae. It is apparent that long before the estimated period of relocation of these mitochondrial genes, the cytosol had lost the splicing ability for group II introns. Therefore, at least eight introns located in the mature protein coding region cannot be the direct descendant of group II introns. Here, we conclude that the presence of these introns is due to the invasion of spliceosomal introns, which occurred during the evolution of Chlorophyceae. This finding provides concrete evidence supporting the "intron-late" model, which rests largely on the mobility of spliceosomal introns. PMID:11675593

  12. Compendium of models from a gauge U(1) framework

    NASA Astrophysics Data System (ADS)

    Ma, Ernest

    2016-06-01

    A gauge U(1) framework was established in 2002 to extend the supersymmetric Standard Model. It has many possible realizations. Whereas all have the necessary and sufficient ingredients to explain the possible 750 GeV diphoton excess, observed recently by the ATLAS and CMS Collaborations at the large hadron collider (LHC), they differ in other essential aspects. A compendium of such models is discussed.

  13. Underground storage tank 431-D1U1, Closure Plan

    SciTech Connect

    Mancieri, S.

    1993-09-01

    This document contains information about the decommissioning of Tank 431-D1U1. This tank was installed in 1965 for diesel fuel storage. This tank will remain in active usage until closure procedures begin. Soils and ground water around the tank will be sampled to check for leakage. Appendices include; proof of proper training for workers, health and safety briefing record, task hazard analysis summary, and emergency plans.

  14. Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly.

    PubMed

    Wu, Guowei; Adachi, Hironori; Ge, Junhui; Stephenson, David; Query, Charles C; Yu, Yi-Tao

    2016-03-15

    Pseudouridine (Ψ) is the most abundant internal modification identified in RNA, and yet little is understood of its effects on downstream reactions. Yeast U2 snRNA contains three conserved Ψs (Ψ35, Ψ42, and Ψ44) in the branch site recognition region (BSRR), which base pairs with the pre-mRNA branch site during splicing. Here, we show that blocks to pseudouridylation at these positions reduce the efficiency of pre-mRNA splicing, leading to growth-deficient phenotypes. Restoration of pseudouridylation at these positions using designer snoRNAs results in near complete rescue of splicing and cell growth. These Ψs interact genetically with Prp5, an RNA-dependent ATPase involved in monitoring the U2 BSRR-branch site base-pairing interaction. Biochemical analysis indicates that Prp5 has reduced affinity for U2 snRNA that lacks Ψ42 and Ψ44 and that Prp5 ATPase activity is reduced when stimulated by U2 lacking Ψ42 or Ψ44 relative to wild type, resulting in inefficient spliceosome assembly. Furthermore, in vivo DMS probing analysis reveals that pseudouridylated U2, compared to U2 lacking Ψ42 and Ψ44, adopts a slightly different structure in the branch site recognition region. Taken together, our results indicate that the Ψs in U2 snRNA contribute to pre-mRNA splicing by directly altering the binding/ATPase activity of Prp5. PMID:26873591

  15. A dominant negative mutation in the conserved RNA helicase motif 'SAT' causes splicing factor PRP2 to stall in spliceosomes.

    PubMed Central

    Plumpton, M; McGarvey, M; Beggs, J D

    1994-01-01

    To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibited cell growth by causing a defect in pre-mRNA splicing. This defect was partially alleviated by simultaneous co-overexpression of wild-type PRP2. The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes. Immunoprecipitation with anti-PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remained associated. The PRP2-dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding. Purified dominant negative PRP2 protein had approximately 40% of the wild-type level of RNA-stimulated ATPase activity. As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein. Images PMID:8112301

  16. Lepton-Flavor Violating Signatures in Supersymmetric U(1)' Seesaw

    NASA Astrophysics Data System (ADS)

    Chun, Eung Jin

    In a supersymmetric U(1)' seesaw model, a right-handed sneutrino can be a good thermal dark matter candidate if the extra gaugino tilde{Z}^{prime} is light enough to provide an appropriate annihilation cross-section through a t-channel diagram. We first discuss how right thermal relic density of the right-handed sneutrino dark matter can arise and then explore lepton number and flavor violating signatures followed by cascade production of tilde{Z}^{prime} from the third generation squarks at the LHC.

  17. Higgs Bosons in the NMSSM and its U(1) Extensions

    SciTech Connect

    Gunion, John F.

    2008-11-23

    I specify the characteristics of a Higgs boson that would be 'ideal' in the light of current data and theoretical attractiveness. I then review why it is that the Higgs bosons of the Standard Model and the Minimal Supersymmetric Model cannot be ideal whereas the lightest Higgs boson of the Next to Minimal Supersymmetric Model can be ideal. Experimental consequences for Higgs and supersymmetry discovery are then reviewed. I then examine the alternatives to the NMSSM in which the MSSM is extended via an extra U(1) symmetry.

  18. U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP.

    PubMed

    Vickers, Timothy A; Sabripour, Mahyar; Crooke, Stanley T

    2011-05-01

    U1 Adaptors are a recently reported novel approach for targeted reduction of mRNA transcripts. A U1 adaptor oligonucleotide comprising of a target-complimentary hybridization domain and a U1 recruitment domain, directs the U1 snRNP complex to the terminal exon of a targeted gene, subsequently inhibiting poly(A) tail addition and leading to degradation of that RNA species within the nucleus. Here, we present data demonstrating U1 adapter-mediated gene silencing can result in significant 'off-target' silencing effects as demonstrated by the reduction of multiple mRNA species that were not intended to be targeted. Our data suggest that a substantial portion of this U1 adaptor-mediated off-target mRNA reduction is the result of sequestration U1 snRNP at levels sufficient to affect splicing and processing of non-target transcripts. PMID:21415007

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

    SciTech Connect

    Gesnel, Marie-Claude; Theoleyre, Sandrine; Del Gatto-Konczak, Fabienne; Breathnach, Richard . E-mail: breathna@nantes.inserm.fr

    2007-07-13

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

  20. A quantum defect analysis of heavy Rydberg behaviour in the B(u+1Σ) and B″ B ‾ (u+1Σ) states of H2 and the B(u+1Σ) state of D2

    NASA Astrophysics Data System (ADS)

    Lawley, Kenneth P.; Donovan, Robert J.

    2016-08-01

    Heavy Rydberg behaviour in the B(u+1Σ) and B″ B ‾ (u+1Σ) ion-pair states of H2 and the B(u+1Σ) state of D2, is analysed in terms of the absolute quantum defects of the vibronic levels. The influence of the inner repulsive wall of ion-pair potentials on heavy Rydberg behaviour is considered and shown to determine the size of both absolute quantum defects and their energy dependence.

  1. Regularized path integrals and anomalies: U(1) chiral gauge theory

    NASA Astrophysics Data System (ADS)

    Kopper, Christoph; Lévêque, Benjamin

    2012-02-01

    We analyze the origin of the Adler-Bell-Jackiw anomaly of chiral U(1) gauge theory within the framework of regularized path integrals. Momentum or position space regulators allow for mathematically well-defined path integrals but violate local gauge symmetry. It is known how (nonanomalous) gauge symmetry can be recovered in the renormalized theory in this case [Kopper, C. and Müller, V. F., "Renormalization of spontaneously broken SU(2) Yang-Mills theory with flow equations," Rev. Math. Phys. 21, 781 (2009)], 10.1142/S0129055X0900375X. Here we analyze U(1) chiral gauge theory to show how the appearance of anomalies manifests itself in such a context. We show that the three-photon amplitude leads to a violation of the Slavnov-Taylor identities which cannot be restored on taking the UV limit in the renormalized theory. We point out that this fact is related to the nonanalyticity of this amplitude in the infrared region.

  2. Wilson loops in noncompact U(1) gauge theories at criticality

    SciTech Connect

    Metlitski, Max A.

    2008-04-15

    We study the properties of Wilson loops in three-dimensional noncompact U(1) gauge theories with global Abelian symmetries. We use duality in the continuum and on the lattice to argue that, close to the critical point between the Higgs and Coulomb phases, all correlators of the Wilson loops are periodic functions of the Wilson loop charge, Q. The period depends on the global symmetry of the theory, which determines the magnetic flux carried by the dual particles. For single flavor scalar electrodynamics, the emergent period is Q=1. In the general case of N complex scalars with a U(1){sup N-1} global symmetry, the period is Q=N. We also give some arguments why this phenomenon does not generalize to theories with a full non-Abelian SU(N) symmetry, where no periodicity in Q is expected. Implications for lattice simulations, as well as for physical systems, such as easy-plane antiferromagnets and disordered superfluids, are noted.

  3. SU(2) x U(1) vacuum and the Centauro events

    NASA Technical Reports Server (NTRS)

    Kazanas, D.; Balasubrahmanyan, V. K.; Streitmatter, R. E.

    1985-01-01

    It is proposed that the fireballs invoked to explain the Centauro events are bubbles of a metastable superdense state of nuclear matter, created in high energy (E approximately 10 to the 15th power eV) cosmic ray collisions at the top of the atmosphere. If these bubbles are created with a Lorentz factor gamma approximately equals 10 at their CM frame, the objections against the origin of these events in cosmic ray interactions are overcome. A relationship then between their lifetime, tau, and the threshold energy for bubble formation, E sub th, appears to be insensitive to the value of tau and always close to E sub th approximately 10 to 15th power eV. Finally it is speculated that these bubbles might be manifestations of the SU(2) x U(1) false vacuum excited in these collisions. The absence of in the Centauro events is then explained by the decay modes of these excitations.

  4. U(1) prime dark matter and R-parity violation

    SciTech Connect

    Brahm, D.E.

    1990-04-01

    Attempts to understand physics beyond the Standard Model must face many phenomenological constraint, from recent Z{sup {degree}} data, neutral current measurements, cosmology and astrophysics, neutrino experiments, tests of lepton-and baryon-number conservation and CP violation, and many other ongoing experiments. The most interesting models are those which are allowed by current data, but offer predictions which can soon be experimentally confirmed or refuted. Two classes of such models are explored in this dissertation. The first, containing an extra U(1){prime} gauge group, has a dark matter candidate which could soon be detected. The second, incorporating supersymmetry with R-parity violation, predicts rare Z{sup {degree}} decays at LEP; some of these models can already be ruled out by LEP data and gluino searches at the Tevatron. 54 refs., 31 figs.

  5. Multivalent binding of formin-binding protein 21 (FBP21)-tandem-WW domains fosters protein recognition in the pre-spliceosome.

    PubMed

    Klippel, Stefan; Wieczorek, Marek; Schümann, Michael; Krause, Eberhard; Marg, Berenice; Seidel, Thorsten; Meyer, Tim; Knapp, Ernst-Walter; Freund, Christian

    2011-11-01

    The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome. PMID:21917930

  6. Aggregation Properties of the Small Nuclear Ribonucleoprotein U1-70K in Alzheimer Disease*

    PubMed Central

    Diner, Ian; Hales, Chadwick M.; Bishof, Isaac; Rabenold, Lake; Duong, Duc M.; Yi, Hong; Laur, Oskar; Gearing, Marla; Troncoso, Juan; Thambisetty, Madhav; Lah, James J.; Levey, Allan I.; Seyfried, Nicholas T.

    2014-01-01

    Recent evidence indicates that U1-70K and other U1 small nuclear ribonucleoproteins are Sarkosyl-insoluble and associate with Tau neurofibrillary tangles selectively in Alzheimer disease (AD). Currently, the mechanisms underlying the conversion of soluble nuclear U1 small nuclear ribonucleoproteins into insoluble cytoplasmic aggregates remain elusive. Based on the biochemical and subcellular distribution properties of U1-70K in AD, we hypothesized that aggregated U1-70K itself or other biopolymers (e.g. proteins or nucleic acids) interact with and sequester natively folded soluble U1-70K into insoluble aggregates. Here, we demonstrate that total homogenates from AD brain induce soluble U1-70K from control brain or recombinant U1-70K to become Sarkosyl-insoluble. This effect was not dependent on RNA and did not correlate with detergent-insoluble Tau levels as AD homogenates with reduced levels of these components were still capable of inducing U1-70K aggregation. In contrast, proteinase K-treated AD homogenates and Sarkosyl-soluble AD fractions were unable to induce U1-70K aggregation, indicating that aggregated proteins in AD brain are responsible for inducing soluble U1-70K aggregation. It was determined that the C terminus of U1-70K, which harbors two disordered low complexity (LC) domains, is necessary for U1-70K aggregation. Moreover, both LC1 and LC2 domains were sufficient for aggregation. Finally, protein cross-linking and mass spectrometry studies demonstrated that a U1-70K fragment harboring the LC1 domain directly interacts with aggregated U1-70K in AD brain. Our results support a hypothesis that aberrant forms of U1-70K in AD can directly sequester soluble forms of U1-70K into insoluble aggregates. PMID:25355317

  7. Molecular architecture of the human U4/U6.U5 tri-snRNP.

    PubMed

    Agafonov, Dmitry E; Kastner, Berthold; Dybkov, Olexandr; Hofele, Romina V; Liu, Wen-Ti; Urlaub, Henning; Lührmann, Reinhard; Stark, Holger

    2016-03-25

    The U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) is a major spliceosome building block. We obtained a three-dimensional structure of the 1.8-megadalton human tri-snRNP at a resolution of 7 angstroms using single-particle cryo-electron microscopy (cryo-EM). We fit all known high-resolution structures of tri-snRNP components into the EM density map and validated them by protein cross-linking. Our model reveals how the spatial organization of Brr2 RNA helicase prevents premature U4/U6 RNA unwinding in isolated human tri-snRNPs and how the ubiquitin C-terminal hydrolase-like protein Sad1 likely tethers the helicase Brr2 to its preactivation position. Comparison of our model with cryo-EM three-dimensional structures of the Saccharomyces cerevisiae tri-snRNP and Schizosaccharomyces pombe spliceosome indicates that Brr2 undergoes a marked conformational change during spliceosome activation, and that the scaffolding protein Prp8 is also rearranged to accommodate the spliceosome's catalytic RNA network. PMID:26912367

  8. SU(2) x U(1) vacuum and the Centauro events

    NASA Technical Reports Server (NTRS)

    Kazanas, D.; Balasubrahmanyan, V. K.; Streitmatter, R. E.

    1984-01-01

    It is proposed that the fireballs invoked to explain the Centauro events are bubbles of a metastable superdense state of nuclear matter, created in high energy (E is approximately 10 to the 15th power eV) cosmic ray collisions at the top of the atmosphere. If these bubbles are created with a Lorentz factor gamma approximately = 10 at their CM frame, the objections against the origin of these events in cosmic ray interactions are overcome. Assuming further, that the Centauro events are to the explosive decay of these metastable bubbles, a relationship between their lifetime, tau, and the threshold energy for bubble formation, E sub th, is derived. The minimum lifetime consistent with such an interpretation in tau is approximately 10 to the -8th power sec, while the E sub th appears to be insensitive to the value of tau and always close to E sub th is approximately 10 to the 15th power eV. Finally it is speculated that if the available CM energy is thermalized in such collisions, these bubbles might be manifestations of excitations of the SU(2) x U(1) false vacuum. The absence of neutral pions in the Centauro events is then explained by the decay of these excitations.

  9. Maneuverability Investigation of an O3U-1 Observation Airplane

    NASA Technical Reports Server (NTRS)

    Thompson, F L; Kirschbaum, H W

    1934-01-01

    This report presents the results of maneuverability tests of an O3U-1 observation airplane. This investigation is the third in a series of similar investigations requested by the Bureau of Aeronautics (Navy) for the purpose of comparing the maneuverability of different airplane types and to provide quantitative data for use in establishing a criterion or method for rating the maneuverability of an airplane. The two former investigations were conducted with the fighter types designated F6C-3 and F6C-4 and have been reported previously. Measurement of the air speed, the angular velocity, the linear acceleration, and positions of the controls were made during abrupt single-control maneuvers with three stop positions for each control, during steady horizontal turns for the determination of minimum radius, and during 180 degree turns by various methods. Flight-path coordinates in two dimensions were determined for the 180 degree turns by means of a special camera obscura designed for the previous investigation of the F6C-4 airplane. All maneuvers were performed at an altitude of approximately 3,000 feet.

  10. Attractive inverse square potential, U(1) gauge, and winding transitions.

    PubMed

    Nisoli, Cristiano; Bishop, A R

    2014-02-21

    The inverse square potential arises in a variety of different quantum phenomena, yet notoriously it must be handled with care: it suffers from pathologies rooted in the mathematical foundations of quantum mechanics. We show that its recently studied conformality breaking corresponds to an infinitely smooth winding-unwinding topological transition for the classical statistical mechanics of a one-dimensional system: this describes the tangling or untangling of floppy polymers under a biasing torque. When the ratio between torque and temperature exceeds a critical value the polymer undergoes tangled oscillations, with an extensive winding number. At lower torque or higher temperature the winding number per unit length is zero. Approaching criticality, the correlation length of the order parameter-the extensive winding number-follows a Kosterlitz-Thouless-type law. The model is described by the Wilson line of a (0+1) U(1) gauge theory, and applies to the tangling or untangling of floppy polymers and to the winding or diffusing kinetics in diffusion-convection reactions. PMID:24579570

  11. Attractive Inverse Square Potential, U(1) Gauge, and Winding Transitions

    NASA Astrophysics Data System (ADS)

    Nisoli, Cristiano; Bishop, A. R.

    2014-02-01

    The inverse square potential arises in a variety of different quantum phenomena, yet notoriously it must be handled with care: it suffers from pathologies rooted in the mathematical foundations of quantum mechanics. We show that its recently studied conformality breaking corresponds to an infinitely smooth winding-unwinding topological transition for the classical statistical mechanics of a one-dimensional system: this describes the tangling or untangling of floppy polymers under a biasing torque. When the ratio between torque and temperature exceeds a critical value the polymer undergoes tangled oscillations, with an extensive winding number. At lower torque or higher temperature the winding number per unit length is zero. Approaching criticality, the correlation length of the order parameter—the extensive winding number—follows a Kosterlitz-Thouless-type law. The model is described by the Wilson line of a (0+1) U(1) gauge theory, and applies to the tangling or untangling of floppy polymers and to the winding or diffusing kinetics in diffusion-convection reactions.

  12. U(1) × SU(2) from the tangent bundle

    NASA Astrophysics Data System (ADS)

    Vargas, Jose G.

    2013-11-01

    Élie Cartan developed modern differential geometry as theory of moving frames. Particles do not enter the equations of structure and thus play a less fundamental role. A Kaluza-Klein (KK) space without compactification brings particles into the core of geometry by making propertime (τ) the fifth dimension. (xi, τ) emerges as the subspace for the quantum sector. This KK space does not make sense in SR, by virtue of non-orthogonality of τ to 3-space (actually), which brings a preferred frame to the fore. In contrast, propertime is perpendicular to 3-space in the "para-Lorentzian structure" with absolute time dilation (PL). Its (xi,τ) subspace looks very much like (t,xi) in SR. And its (t,xi) sector is not made of orthogonal frames but does not cause contradictions with SR, which supporters of the thesis of conventionality of synchronizations have been claiming for many decades. In PL, the conjunction of the Clifford algebras of differential forms (Kaehler's) and of their valuedness gives rise to a commutative algebra of primitive idempotents that embodies the U(1) × SU(2) symmetry. SU(3) also emerges in the process, but we do not deal with this issue beyond proposing the geometric palet of quarks.

  13. Identification of a novel nuclear localization signal and speckle-targeting sequence of tuftelin-interacting protein 11, a splicing factor involved in spliceosome disassembly

    SciTech Connect

    Tannukit, Sissada; Crabb, Tara L.; Hertel, Klemens J.; Wen, Xin; Jans, David A.; Paine, Michael L.

    2009-12-18

    Tuftelin-interacting protein 11 (TFIP11) is a protein component of the spliceosome complex that promotes the release of the lariat-intron during late-stage splicing through a direct recruitment and interaction with DHX15/PRP43. Expression of TFIP11 is essential for cell and organismal survival. TFIP11 contains a G-patch domain, a signature motif of RNA-processing proteins that is responsible for TFIP11-DHX15 interactions. No other functional domains within TFIP11 have been described. TFIP11 is localized to distinct speckled regions within the cell nucleus, although excluded from the nucleolus. In this study sequential C-terminal deletions and mutational analyses have identified two novel protein elements in mouse TFIP11. The first domain covers amino acids 701-706 (VKDKFN) and is an atypical nuclear localization signal (NLS). The second domain is contained within amino acids 711-735 and defines TFIP11's distinct speckled nuclear localization. The identification of a novel TFIP11 nuclear speckle-targeting sequence (TFIP11-STS) suggests that this domain directly interacts with additional spliceosomal components. These data help define the mechanism of nuclear/nuclear speckle localization of the splicing factor TFIP11, with implications for it's function.

  14. Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions

    PubMed Central

    Thanaraj, T. A.; Clark, Francis

    2001-01-01

    It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recognized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5/U6 snRNAs. However, variability in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG introns (for the first time from any species), and show that while constitutive GC-AG introns do possess strong signals at their donor sites, a large subset of alternative GC-AG introns possess weak consensus sequences at their donor sites. Surprisingly, this subset of alternative isoforms shows strong consensus at acceptor exon positions 1 and 2. The improved consensus at the acceptor exon can facilitate a strong interaction with U5 snRNA, which tethers the two exons for ligation during the second step of splicing. Further, these isoforms nearly always possess alternative acceptor sites and exhibit particularly weak polypyrimidine tracts characteristic of AG-dependent introns. The acceptor exon nucleotides are part of the consensus required for the U2AF35-mediated recognition of AG in such introns. Such improved consensus at acceptor exons is not found in either normal or alternative GT-AG introns having weak donor sites or weak polypyrimidine tracts. The changes probably reflect mechanisms that allow GC-AG alternative intron isoforms to cope with two conflicting requirements, namely an apparent need for differential splice strength to direct the choice of alternative sites and a need for improved donor signals to compensate for the central mismatch base pair (C-A) in the RNA duplex of U1 snRNA and the pre-mRNA. The other important findings include (i) one in every twenty alternative introns is a GC-AG intron, and (ii) three of every five observed GC-AG introns are alternative isoforms. PMID:11410667

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

    PubMed Central

    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

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

    PubMed

    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

  17. Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry

    PubMed Central

    Ajuh, Paul; Kuster, Bernhard; Panov, Kostya; Zomerdijk, Joost C.B.M.; Mann, Matthias; Lamond, Angus I.

    2000-01-01

    Recently, we identified proteins that co-purify with the human spliceosome using mass spectrometry. One of the identified proteins, CDC5L, corresponds to the human homologue of the Schizosaccharomyces pombe CDC5+ gene product. Here we show that CDC5L is part of a larger multiprotein complex in HeLa nuclear extract that incorporates into the spliceosome in an ATP-dependent step. We also show that this complex is required for the second catalytic step of pre-mRNA splicing. Immunodepletion of the CDC5L complex from HeLa nuclear extract inhibits the formation of pre-mRNA splicing products in vitro but does not prevent spliceosome assembly. The first catalytic step of pre-mRNA splicing is less affected by immunodepleting the complex. The purified CDC5L complex in HeLa nuclear extract restores pre-mRNA splicing activity when added to extracts that have been immunodepleted using anti-CDC5L antibodies. Using mass spectrometry and database searches, the major protein components of the CDC5L complex have been identified. This work reports a first purification and characterization of a functional, human non-snRNA spliceosome subunit containing CDC5L and at least five additional protein factors. PMID:11101529

  18. A 62,000 molecular weight spliceosome protein crosslinks to the intron polypyrimidine tract.

    PubMed

    Wang, J; Pederson, T

    1990-10-25

    Incubation in HeLa nuclear extract of a 32P-labeled 61 nucleotide-long RNA corresponding to the lariat branch site/polypyrimidine tract/3' splice site of the first intron of human beta-globin pre-mRNA led to the crosslinking of a single protein of approximately 62,000 mol. wt. (p62). p62 corresponds to a polypyrimidine tract-binding protein recently described by Garcia-Blanco et al. (Genes & Dev. 3: 1874-1886, 1989). Crosslinking of p62 to the 61 nt RNA was highly sequence specific. No p62 crosslinking was observed with a 60 nt pGEM vector RNA, a 63 nt RNA antisense to the 61-mer or a 72 nt U2 RNA sequence. p62 crosslinking to the 61 nt RNA was competed by unlabeled 61 nt RNA, by beta-globin pre-mRNA containing intron 1, and by poly(U) and poly(C), but was competed to a lesser extent or not at all by pGEM RNA, a beta-globin RNA lacking intron 1, or poly(A). Experiments with mutated RNAs revealed that neither the lariat branch site adenosine nor the 3' splice site were required for p62 crosslinking to polypyrimidine tract-containing RNA. Elimination of the polypyrimidine tract reduced p62 crosslinking, as did mutation of a polypyrimidine tract UU dinucleotide to GA. However, replacement of a pyrimidine-rich tract immediately adjacent (3') to the lariat branch site with a 57% A + G pGEM vector RNA sequence also significantly reduced p62 crosslinking, indicating the involvement of both this pyrimidine-rich region and the classical polypyrimidine tract adjacent to the 3' splice site. The sites of protein interaction were further defined by RNase H protection experiments, the results of which confirmed the patterns of p62 crosslinking to mutant RNAs. p62 crosslinking was efficiently competed by a DNA oligonucleotide having the same sequence as the 61 nt RNA, showing that p62 requires neither ribose 2' OH groups nor uracil bases for its interaction with the polypyrimidine tract. p62 was not crosslinked to double-stranded 61 nt RNA. Q-Sepharose chromatography of He

  19. Climbing the vertebrate branch of U1A/U2B″ protein evolution

    PubMed Central

    Delaney, Kimberly J.; Williams, Sandra G.; Lawler, Mariah; Hall, Kathleen B.

    2014-01-01

    In the vertebrate lineage of the U1A/U2B″/SNF protein family, the U1A and U2B″ proteins bind to RNA stem–loops in the U1 or U2 snRNPs, respectively. However, their specialization is fairly recent, as they evolved from a single ancestral protein. The progress of their specialization (subfunctionalization) can be monitored by the amino acid sequence changes that give rise to their modern RNA-binding specificity. Using ancestral sequence reconstruction to predict the intermediates on the evolutionary branch, a probable path of sequential changes is defined for U1A and U2B″. The RNA-binding affinity for U1A/U2B″ protein ancestors was measured using modern U1 and U2 snRNA stem–loops and RNA stem–loop variants to understand how the proteins’ RNA specificities evolved. PMID:24840944

  20. Single field inflation in supergravity with a U(1) gauge symmetry

    SciTech Connect

    Heurtier, L.; Khalil, S.; Moursy, A.

    2015-10-19

    A single field inflation based on a supergravity model with a shift symmetry and U(1) extension of the MSSM is analyzed. We show that one of the real components of the two U(1) charged scalar fields plays the role of inflaton with an effective scalar potential similar to the “new chaotic inflation” scenario. Both non-anomalous and anomalous (with Fayet-Iliopoulos term) U(1) are studied. We show that the non-anomalous U(1) scenario is consistent with data of the cosmic microwave background and recent astrophysical measurements. A possible kinetic mixing between U(1) and U(1){sub B−L} is considered in order to allow for natural decay channels of the inflaton, leading to a reheating epoch. Upper limits on the reheating temperature thus turn out to favour an intermediate (∼O(10{sup 13}) GeV) scale B−L symmetry breaking.

  1. Spin Tests of 1/20-Scale Models of the Chance Vought Revised XF6U-1 and F6U-1 Airplanes, TED No. NACA 2390

    NASA Technical Reports Server (NTRS)

    Klinar, Walter J.; Berman, Theodore

    1948-01-01

    An investigation has been conducted in the Langley 20-foot free-spinning tunnel on the 1/20-scale model of the Chance Vought XF6U-1 airplane altered to represent the XF6U-1 airplane as it will be spin-tested in flight, and also altered to represent the F6U-1 airplane as it will be produced for service use. Spin tests were made to determine the effects of control settings and movements at the normal loading. The results show that the spins obtained on the revised XF6U-1 airplane will be oscillatory in roll and yaw and that recoveries by rudder reversal will be rapid. Model test results indicate that the F6U-1 airplane will probably not spin. Inasmuch as the results of this investigation show that the new designs are as good as or better than the original XF6U-1 design in regard to spin recovery, it is felt that the conclusions and recommendations reached for the original design can be applied to the new designs for all loading conditions.

  2. General U(1U(1) F-theory compactifications and beyond: geometry of unHiggsings and novel matter structure

    NASA Astrophysics Data System (ADS)

    Cvetič, Mirjam; Klevers, Denis; Piragua, Hernan; Taylor, Washington

    2015-11-01

    We construct the general form of an F-theory compactification with two U(1) factors based on a general elliptically fibered Calabi-Yau manifold with Mordell-Weil group of rank two. This construction produces broad classes of models with diverse matter spectra, including many that are not realized in earlier F-theory constructions with U(1U(1) gauge symmetry. Generic U(1U(1) models can be related to a Higgsed non-Abelian model with gauge group SU(2)×SU(2)×SU(3), SU(2)3×SU(3), or a subgroup thereof. The nonlocal horizontal divisors of the Mordell-Weil group are replaced with local vertical divisors associated with the Cartan generators of non-Abelian gauge groups from Kodaira singularities. We give a global resolution of codimension two singularities of the Abelian model; we identify the full anomaly free matter content, and match it to the unHiggsed non-Abelian model. The non-Abelian Weierstrass model exhibits a new algebraic description of the singularities in the fibration that results in the first explicit construction of matter in the symmetric representation of SU(3). This matter is realized on double point singularities of the discriminant locus. The construction suggests a generalization to U(1) k factors with k > 2, which can be studied by Higgsing theories with larger non-Abelian gauge groups.

  3. An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses

    PubMed Central

    Schreiber, Claire A.; Sakuma, Toshie; Izumiya, Yoshihiro; Holditch, Sara J.; Hickey, Raymond D.; Bressin, Robert K.; Basu, Upamanyu; Koide, Kazunori; Asokan, Aravind; Ikeda, Yasuhiro

    2015-01-01

    Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors. PMID:26244496

  4. Phenomenological study on the wino radiative decay in anomalous U(1){sup '} models

    SciTech Connect

    Fucito, Francesco; Lionetto, Andrea; Pacifici, Daniel Ricci; Racioppi, Antonio

    2010-12-01

    An extension of the standard model by at least one extra U(1) gauge symmetry has been investigated by many authors. In this paper we explore the possibility that this extra U(1) is anomalous. One possible signature of this model could be given by the photons produced in the decays of the next to lightest supersymmetric particle into the lightest supersymmetric particle.

  5. 46 CFR 54.01-10 - Steam-generating pressure vessels (modifies U-1(g)).

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 2 2014-10-01 2014-10-01 false Steam-generating pressure vessels (modifies U-1(g)). 54... ENGINEERING PRESSURE VESSELS General Requirements § 54.01-10 Steam-generating pressure vessels (modifies U-1(g)). (a) Pressure vessels in which steam is generated are classed as “Unfired Steam Boilers” except...

  6. 46 CFR 54.01-10 - Steam-generating pressure vessels (modifies U-1(g)).

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 2 2012-10-01 2012-10-01 false Steam-generating pressure vessels (modifies U-1(g)). 54... ENGINEERING PRESSURE VESSELS General Requirements § 54.01-10 Steam-generating pressure vessels (modifies U-1(g)). (a) Pressure vessels in which steam is generated are classed as “Unfired Steam Boilers” except...

  7. 46 CFR 54.01-10 - Steam-generating pressure vessels (modifies U-1(g)).

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 2 2013-10-01 2013-10-01 false Steam-generating pressure vessels (modifies U-1(g)). 54... ENGINEERING PRESSURE VESSELS General Requirements § 54.01-10 Steam-generating pressure vessels (modifies U-1(g)). (a) Pressure vessels in which steam is generated are classed as “Unfired Steam Boilers” except...

  8. Integrated Approaches for Analyzing U1-70K Cleavage in Alzheimer’s Disease

    PubMed Central

    2015-01-01

    The accumulation of pathologic protein fragments is common in neurodegenerative disorders. We have recently identified in Alzheimer’s disease (AD) the aggregation of the U1-70K splicing factor and abnormal RNA processing. Here, we present that U1-70K can be cleaved into an N-terminal truncation (N40K) in ∼50% of AD cases, and the N40K abundance is inversely proportional to the total level of U1-70K. To map the cleavage site, we compared tryptic peptides of N40K and stable isotope labeled U1-70K by liquid chromatography–tandem mass spectrometry (MS), revealing that the proteolysis site is located in a highly repetitive and hydrophilic domain of U1-70K. We then adapted Western blotting to map the cleavage site in two steps: (i) mass spectrometric analysis revealing that U1-70K and N40K share the same N-termini and contain no major modifications; (ii) matching N40K with a series of six recombinant U1-70K truncations to define the cleavage site within a small region (Arg300 ± 6 residues). Finally, N40K expression led to substantial degeneration of rat primary hippocampal neurons. In summary, we combined multiple approaches to identify the U1-70K proteolytic site and found that the N40K fragment might contribute to neuronal toxicity in Alzheimer’s disease. PMID:24902715

  9. Closure report for underground storage tank 141-R3U1 and its associated underground piping

    SciTech Connect

    Mallon, B.J.; Blake, R.G.

    1994-03-01

    Underground storage tank UST 141-R3U1 at Lawrence Livermore National Laboratory (LLNL), was registered with the State Water Resources Control Board on June 27, 1984. This tank system consisted of a concrete tank, lined with polyvinyl chloride, and approximately 100 feet of PVC underground piping. UST 141-R3U1 had a capacity of 450 gallons. The underground piping connected three floor drains and one sink inside Building 141 to UST 141-R3U1. The wastewater collected in UST 141-R3U1 contained organic solvents, metals, and inorganic acids. On November 30, 1987, the 141-R3U1 tank system failed a precision tank test. The 141-R3U1 tank system was subsequently emptied and removed from service pending further precision tests to determine the location of the leak within the tank system. A precision tank test on February 5, 1988, was performed to confirm the November 30, 1987 test. Four additional precision tests were performed on this tank system between February 25, 1988, and March 6, 1988. The leak was located where the inlet piping from Building 141 penetrates the concrete side of UST 141-R3U1. The volume of wastewater that entered the backfill and soil around and/or beneath UST 141-R3U1 is unknown. On December 13, 1989, the LLNL Environmental Restoration Division submitted a plan to close UST 141-R3U1 and its associated piping to the Alameda County Department of Environmental Health. UST 141-R3U1 was closed as an UST, and shall be used instead as additional secondary containment for two aboveground storage tanks.

  10. U1 small nuclear ribonucleoproteins (snRNPs) aggregate in Alzheimer’s disease due to autosomal dominant genetic mutations and trisomy 21

    PubMed Central

    2014-01-01

    Background We recently identified U1 small nuclear ribonucleoprotein (snRNP) tangle-like aggregates and RNA splicing abnormalities in sporadic Alzheimer’s disease (AD). However little is known about snRNP biology in early onset AD due to autosomal dominant genetic mutations or trisomy 21 in Down syndrome. Therefore we investigated snRNP biochemical and pathologic features in these disorders. Findings We performed quantitative proteomics and immunohistochemistry in postmortem brain from genetic AD cases. Electron microscopy was used to characterize ultrastructural features of pathologic aggregates. U1-70k and other snRNPs were biochemically enriched in the insoluble fraction of human brain from subjects with presenilin 1 (PS1) mutations. Aggregates of U1 snRNP-immunoreactivity formed cytoplasmic tangle-like structures in cortex of AD subjects with PS1 and amyloid precursor protein (APP) mutations as well as trisomy 21. Ultrastructural analysis with electron microscopy in an APP mutation case demonstrated snRNP immunogold labeling of paired helical filaments (PHF). Conclusions These studies identify U1 snRNP pathologic changes in brain of early onset genetic forms of AD. Since dominant genetic mutations and trisomy 21 result in dysfunctional amyloid processing, the findings suggest that aberrant β-amyloid processing may influence U1 snRNP aggregate formation. PMID:24773620

  11. Functional analysis of U1-70K interacting SR proteins in pre-mRNA splicing in Arabidopsis

    SciTech Connect

    A.S.N. Reddy

    2008-11-25

    Proteins of a serine/arginine-rich (SR) family are part of the spliceosome and are implicated in both constitutive and alternative splicing of pre-mRNAs. With the funding from DOE we have been studying alternative of splicing of genes encoding serine/arginine-rich (SR) proteins and the roles of SR proteins that interact with U1-70K in regulating basic and alternative splicing. Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins and its regulation by hormones and stresses: We analyzed the splicing of all 19 Arabidopsis genes in different tissues, during different seedling stages and in response to various hormonal and stress treatments. Remarkably, about 90 different transcripts are produced from 15 SR genes, thereby increasing the transcriptome complexity of SR genes by about five fold. Using the RNA isolated from polysomes we have shown that most of the splice variants are recruited for translation. Alternative splicing of some SR genes is controlled in a developmental and tissue-specific manner (Palusa et al., 2007). Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) and ultraviolet light dramatically altered alternative splicing of pre-mRNAs of several SR genes whereas hormones altered the splicing of only two SR genes (Palusa et al., 2007). Localization and dynamics of a novel serine/arginine-rich protein that interacts with U1-70K: We analyzed the intranuclear movement of SR45 fused to GFP by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We demonstrate that the movement of GFP-SR45 is ATP-dependent. Interestingly, inhibition of transcription or phosphorylation slowed the mobility of GFP-SR45 (Ali et al., 2006). Our studies have revealed that the nuclear localization signals are located in arg/ser-rich domains (RS) 1 and 2, whereas the speckle targeting signals are exclusively present in RS2 (Ali et al., 2006). The regulation of

  12. New U(1) gauge model of radiative lepton masses with sterile neutrino and dark matter

    NASA Astrophysics Data System (ADS)

    Adhikari, Rathin; Borah, Debasish; Ma, Ernest

    2016-04-01

    An anomaly-free U(1) gauge extension of the standard model (SM) is presented. Only one Higgs doublet with a nonzero vacuum expectation is required as in the SM. New fermions and scalars as well as all SM particles transform nontrivially under this U(1), resulting in a model of three active neutrinos and one sterile neutrino, all acquiring radiative masses. Charged-lepton masses are also radiative as well as the mixing between active and sterile neutrinos. At the same time, a residual Z2 symmetry of the U(1) gauge symmetry remains exact, allowing for the existence of dark matter.

  13. Shadow Higgs boson from a scale-invariant hidden U(1){sub s} model

    SciTech Connect

    Chang, W.-F.; Ng, John N.; Wu, Jackson M. S.

    2007-06-01

    We study a scale-invariant SU(2)xU(1){sub Y}xU(1){sub s} model which has only dimensionless couplings. The shadow U(1){sub s} is hidden, and it interacts with the standard model (SM) solely through mixing in the scalar sector and kinetic mixing of the U(1) gauge bosons. The gauge symmetries are broken radiatively by the Coleman-Weinberg mechanism. Lifting of the flat direction in the scalar potential gives rise to a light scalar, the scalon, or the shadow Higgs, and a heavier scalar which we identify as the SM Higgs boson. The phenomenology of this model is discussed. In particular, the constraints on the shadow Higgs in different mass ranges, and the possibility of discovering a shadow Higgs with a mass a few tens of GeV in precision t-quark studies at the LHC, are investigated.

  14. 10. DETAILED INTERIOR VIEW OF WEB AT U1L1, SHOWING VERTICAL, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. DETAILED INTERIOR VIEW OF WEB AT U1L1, SHOWING VERTICAL, ARCH RIB WELDED/BOLTED GUSSET PLATE AND GUARDRAIL, LOOKING SOUTH - Cottonville Bridge, County Road D-61 at Farmer's Creek, Maquoketa, Jackson County, IA

  15. Constraints on the U(1)L gauge boson in a wide mass range

    NASA Astrophysics Data System (ADS)

    Jeong, Yu Seon; Kim, C. S.; Lee, Hye-Sung

    2016-04-01

    There is a growing interest for the search of new light gauge bosons. The small mass of a new boson can turn various kinds of low-energy experiments to a new discovery machine, depending on their couplings to the Standard Model particles. It is important to understand the properties of each type of gauge boson and their current constraints for a given mass. While the dark photon (which couples to the electric charges) and the U(1)B‑L gauge boson have been well studied in an extensive mass range, the U(1)L gauge boson has not been fully investigated yet. We consider the gauge boson of the U(1)L in a wide mass range mZ‧≈ 0-1012eV and investigate the constraints on its coupling from various experiments, discussing the similarities and differences from the dark photon and the U(1)B‑L gauge boson.

  16. Dark Matter and neutrino masses from global U(1) B - L symmetry breaking

    NASA Astrophysics Data System (ADS)

    Lindner, Manfred; Schmidt, Daniel; Schwetz, Thomas

    2011-11-01

    We present a scenario where neutrino masses and Dark Matter are related due to a global U(1) B - L symmetry. Specifically we consider neutrino mass generation via the Zee-Babu two-loop mechanism, augmented by a scalar singlet whose VEV breaks the global U(1) B - L symmetry. In order to obtain a Dark Matter candidate we introduce two Standard Model singlet fermions. They form a Dirac particle and are stable because of a remnant Z2 symmetry. Hence, in this model the stability of Dark Matter follows from the global U(1) B - L symmetry. We discuss the Dark Matter phenomenology of the model, and compare it to similar models based on gauged U(1) B - L. We argue that in contrast to the gauged versions, the model based on the global symmetry does not suffer from severe constraints from Z‧ searches.

  17. All exactly solvable U(1)-invariant quantum spin 1 chains from Hecke algebra

    SciTech Connect

    Alcarez, F.C. ); Koberle, R. ); Lima-Santos, A. )

    1992-12-10

    In this paper, the authors obtain all exactly integrable spin 1 quantum chains, which are U(1) invariant and satisfy the Hecke algebra. The authors present various generalizations for arbitrary spin S and discuss their solution via Bethe ansatz methods.

  18. 40 CFR Table U-1 to Subpart U of... - CO2 Emission Factors for Common Carbonates

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 21 2011-07-01 2011-07-01 false CO2 Emission Factors for Common.... 98, Subpt. U, Table U-1 Table U-1 to Subpart U of Part 98—CO2 Emission Factors for Common Carbonates Mineral name—carbonate CO2 emission factor(tons CO2/ton carbonate) Limestone—CaCO3 0.43971...

  19. Classically conformal U (1 )' extended standard model and Higgs vacuum stability

    NASA Astrophysics Data System (ADS)

    Oda, Satsuki; Okada, Nobuchika; Takahashi, Dai-suke

    2015-07-01

    We consider the minimal U (1 )' extension of the standard model (SM) with conformal invariance at the classical level, where in addition to the SM particle contents, three generations of right-handed neutrinos and a U (1 )' Higgs field are introduced. In the presence of the three right-handed neutrinos, which are responsible for the seesaw mechanism, this model is free from all the gauge and gravitational anomalies. The U (1 )' gauge symmetry is radiatively broken via the Coleman-Weinberg mechanism, by which the U (1 )' gauge boson (Z' boson) mass as well as the Majorana mass for the right-handed neutrinos are generated. The radiative U (1 )' symmetry breaking also induces a negative mass squared for the SM Higgs doublet to trigger the electroweak symmetry breaking. In this context, we investigate a possibility to solve the SM Higgs vacuum instability problem. The model includes only three free parameters (U (1 )' charge of the SM Higgs doublet, U (1 )' gauge coupling and Z' boson mass), for which we perform parameter scan, and identify a parameter region resolving the SM Higgs vacuum instability. We also examine naturalness of the model. The heavy states associated with the U (1 )' symmetry breaking contribute to the SM Higgs self-energy. We find an upper bound on Z' boson mass, mZ'≲6 TeV , in order to avoid a fine-tuning severer than 10% level. The Z' boson in this mass range can be discovered at the LHC Run-2 in the near future.

  20. 46 CFR 54.01-5 - Scope (modifies U-1 and U-2).

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 2 2014-10-01 2014-10-01 false Scope (modifies U-1 and U-2). 54.01-5 Section 54.01-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING PRESSURE VESSELS General Requirements § 54.01-5 Scope (modifies U-1 and U-2). (a) This part contains requirements for pressure vessels. table 54.01-5(a) gives...

  1. Hidden gauged U (1 ) model: Unifying scotogenic neutrino and flavor dark matter

    NASA Astrophysics Data System (ADS)

    Yu, Jiang-Hao

    2016-06-01

    In both scotogenic neutrino and flavor dark matter models, the dark sector communicates with the standard model fermions via Yukawa portal couplings. We propose an economic scenario where the scotogenic neutrino and a flavored mediator share the same inert Higgs doublet and all are charged under a hidden gauged U (1 ) symmetry. The dark Z2 symmetry in the dark sector is regarded as the remnant of this hidden U (1 ) symmetry breaking. In particular, we investigate a dark U (1 )D [and also U (1 )B-L] model which unifies the scotogenic neutrino and top-flavored mediator. Thus dark tops and dark neutrinos are the standard model fermion partners, and the dark matter could be the inert Higgs or the lightest dark neutrino. We note that this model has rich collider signatures on dark tops, the inert Higgs and the Z' gauge boson. Moreover, the scalar associated to the U (1 )D [and also U (1 )B -L ] symmetry breaking could explain the 750 GeV diphoton excess reported by ATLAS and CMS recently.

  2. F-theory and all things rational: surveying U(1) symmetries with rational sections

    NASA Astrophysics Data System (ADS)

    Lawrie, Craig; Schäfer-Nameki, Sakura; Wong, Jin-Mann

    2015-09-01

    We study elliptic fibrations for F-theory compactifications realizing 4d and 6d supersymmetric gauge theories with abelian gauge factors. In the fibration these U(1) symmetries are realized in terms of additional rational section. We obtain a universal characterization of all the possible U(1) charges of matter fields by determining the corresponding codimension two fibers with rational sections. In view of modelling supersymmetric Grand Unified Theories, one of the main examples that we analyze are U(1) symmetries for SU(5) gauge theories with overline{5} and 10 matter. We use a combination of constraints on the normal bundle of rational curves in Calabi-Yau three- and four-folds, as well as the splitting of rational curves in the fibers in codimension two, to determine the possible configurations of smooth rational sections. This analysis straightforwardly generalizes to multiple U(1)s. We study the flops of such fibers, as well as some of the Yukawa couplings in codimension three. Furthermore, we carry out a universal study of the U(1)-charged GUT singlets, including their KK-charges, and determine all realizations of singlet fibers. By giving vacuum expectation values to these singlets, we propose a systematic way to analyze the Higgsing of U(1)s to discrete gauge symmetries in F-theory.

  3. Closure report for underground storage tank 161-R1U1 and its associated underground piping

    SciTech Connect

    Mallon, B.J.; Blake, R.G.

    1994-05-01

    Underground storage tank (UST) 161-31 R at the Lawrence Livermore National Laboratory (LLNL) was registered with the State Water Resources Control Board on June 27, 1984. UST 161-31R was subsequently renamed UST 161-R1U1 (Fig. A-1, Appendix A). UST 161-R1U1 was installed in 1976, and had a capacity of 383 gallons. This tank system consisted of a fiberglass reinforced plastic tank, approximately 320 feet of polyvinyl chloride (PVC) underground piping from Building 161, and approximately 40 feet of PVC underground piping from Building 160. The underground piping connected laboratory drains and sinks inside Buildings 160 and 161 to UST 161-R1U1. The wastewater collected in UST 161-R1U1, contained organic solvents, metals, inorganic acids, and radionuclides, most of which was produced within Building 161. On June 28, 1989, the UST 161-R1U1 piping system.around the perimeter of Building 161 failed a precision test performed by Gary Peters Enterprises (Appendix B). The 161-R1U1 tank system was removed from service after the precision test. In July 1989, additional hydrostatic tests and helium leak detection tests were performed (Appendix B) to determine the locations of the piping failures in the Building 161 piping system. The locations of the piping system failures are shown in Figure A-2 (Appendix A). On July 11, 1989, LLNL submitted an Unauthorized Release Report to Alameda County Department of Environmental Health (ACDEH), Appendix C.

  4. Defective control of pre–messenger RNA splicing in human disease

    PubMed Central

    Shkreta, Lulzim

    2016-01-01

    Examples of associations between human disease and defects in pre–messenger RNA splicing/alternative splicing are accumulating. Although many alterations are caused by mutations in splicing signals or regulatory sequence elements, recent studies have noted the disruptive impact of mutated generic spliceosome components and splicing regulatory proteins. This review highlights recent progress in our understanding of how the altered splicing function of RNA-binding proteins contributes to myelodysplastic syndromes, cancer, and neuropathologies. PMID:26728853

  5. The nuclear matrix protein p255 is a highly phosphorylated form of RNA polymerase II largest subunit which associates with spliceosomes.

    PubMed Central

    Vincent, M; Lauriault, P; Dubois, M F; Lavoie, S; Bensaude, O; Chabot, B

    1996-01-01

    The monoclonal antibody CC-3 recognizes a phosphodependent epitope on a 255 kDa nuclear matrix protein (p255) recently shown to associate with splicing complexes as part of the [U4/U6.U5] tri-snRNP particle [Chabot et al. (1995) Nucleic Acids Res. 23, 3206-3213]. In mouse and Drosophila cultured cells the electrophoretic mobility of p255, faster in the latter species, was identical to that of the hyperphosphorylated form of RNA polymerase II largest subunit (IIo). The CC-3 immunoreactivity of p255 was abolished by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, which is known to cause the dephosphorylation of the C-terminal domain of subunit IIo by inhibiting the TFIIH-associated kinase. The identity of p255 was confirmed by showing that CC-3-immunoprecipitated p255 was recognized by POL3/3 and 8WG16, two antibodies specific to RNA polymerase II largest subunit. Lastly, the recovery of RNA polymerase II largest subunit from HeLa splicing mixtures was compromised by EDTA, which prevents the interaction of p255 with splicing complexes and inhibits splicing. Our results indicate that p255 represents a highly phosphorylated form of RNA polymerase II largest subunit physically associated with spliceosomes and possibly involved in coupling transcription to RNA processing. PMID:8972849

  6. Physiologic Expression of Sf3b1(K700E) Causes Impaired Erythropoiesis, Aberrant Splicing, and Sensitivity to Therapeutic Spliceosome Modulation.

    PubMed

    Obeng, Esther A; Chappell, Ryan J; Seiler, Michael; Chen, Michelle C; Campagna, Dean R; Schmidt, Paul J; Schneider, Rebekka K; Lord, Allegra M; Wang, Lili; Gambe, Rutendo G; McConkey, Marie E; Ali, Abdullah M; Raza, Azra; Yu, Lihua; Buonamici, Silvia; Smith, Peter G; Mullally, Ann; Wu, Catherine J; Fleming, Mark D; Ebert, Benjamin L

    2016-09-12

    More than 80% of patients with the refractory anemia with ring sideroblasts subtype of myelodysplastic syndrome (MDS) have mutations in Splicing Factor 3B, Subunit 1 (SF3B1). We generated a conditional knockin mouse model of the most common SF3B1 mutation, Sf3b1(K700E). Sf3b1(K700E) mice develop macrocytic anemia due to a terminal erythroid maturation defect, erythroid dysplasia, and long-term hematopoietic stem cell (LT-HSC) expansion. Sf3b1(K700E) myeloid progenitors and SF3B1-mutant MDS patient samples demonstrate aberrant 3' splice-site selection associated with increased nonsense-mediated decay. Tet2 loss cooperates with Sf3b1(K700E) to cause a more severe erythroid and LT-HSC phenotype. Furthermore, the spliceosome modulator, E7017, selectively kills SF3B1(K700E)-expressing cells. Thus, SF3B1(K700E) expression reflects the phenotype of the mutation in MDS and may be a therapeutic target in MDS. PMID:27622333

  7. Cytochrome P450 107U1 is required for sporulation and antibiotic production in Streptomyces coelicolor

    PubMed Central

    Tian, Zhenghua; Cheng, Qian; Yoshimoto, Francis K.; Lei, Li; Lamb, David C.; Guengerich, F. Peter

    2013-01-01

    The filamentous bacterium Streptomyces coelicolor has a complex life cycle involving the formation of hair-like aerial mycelia on the colony surface, which differentiate into chains of spores. Genes required for the initiation of aerial mycelium formation have been termed ‘bld’ (bald), describing the smooth, undifferentiated colonies of mutant strains. We report the identification of a new bld gene designated as sco3099 and biochemical analysis of its encoded enzyme, cytochrome P450 (P450, or CYP) 107U1. Deletion of sco3099 resulted in a mutant defective in aerial hyphae sporulation and sensitive to heat shock, indicating that P450 107U1 plays a key role in growth and development of S. coelicolor. This is the first P450 reported to participate in a sporulation process in Streptomycetes. The substrate and catalytic properties of P450 107U1 were further investigated in mass spectrometry-based metabolomic studies. Glycocholic acid (from the medium) was identified as a substrate of P450 107U1 and was oxidized to glyco-7-oxo-deoxycholic acid. Although this reaction is apparently not relevant to the observed sporulation deficiency, it suggests that P450 107U1 might exert its physiological function by oxidizing other steroid-like molecules. PMID:23357279

  8. Pigmentary degenerative maculopathy as prominent phenotype in an Italian SPG56/CYP2U1 family.

    PubMed

    Leonardi, Luca; Ziccardi, Lucia; Marcotulli, Christian; Rubegni, Anna; Longobardi, Antonino; Serrao, Mariano; Storti, Eugenia; Pierelli, Francesco; Tessa, Alessandra; Parisi, Vincenzo; Santorelli, Filippo M; Carlo, Casali

    2016-04-01

    SPG56 is an autosomal recessive form of hereditary spastic paraplegia (HSP) associated with mutations in CYP2U1. There is no clear documentation of visual impairment in the few reported cases of SPG56, although this form is complex on clinical ground and visual deficit are extremely frequent in complicated HSP. We report three patients in a consanguineous family harboring the novel homozygous c.1168C>T (p.R390*) in SPG56/CYP2U1, and showing a pigmentary degenerative maculopathy associated with progressive spastic paraplegia. Furthermore, we characterized precisely the ophthalmologic phenotype through indirect ophthalmoscopy, retinal optical coherence tomography and visual evoked potentials. This is the first formal report of pigmentary degenerative maculopathy associated with a CYP2U1 homozygous mutation. PMID:26914923

  9. Nonlinear optical conductivity of U (1 ) spin liquids with large spinon Fermi surfaces

    NASA Astrophysics Data System (ADS)

    Ma, Yuan-Fei; Ng, Tai-Kai

    2016-06-01

    In this paper we study the nonlinear current response of U (1 ) spin liquids with large spinon Fermi surfaces under the perturbation of a time-dependent ac electric field E (t ) within the framework of an effective U (1 ) gauge theory. In particular, the third-order nonlinear current response to ac electric fields is derived. We show that as in the case of linear current response, an in-gap power-law (˜ωη ) response is found for the nonlinear current at low frequency. The nonlinear susceptibility may also induce through process of third harmonic generation propagating EM wave with frequency 3 ω inside the spin liquids.

  10. Modular invariant gaugino condensation in the presence of ananomalous U(1)*

    SciTech Connect

    Gaillard, Mary K.; Giedt, Joel; Mints, Aleksey L.

    2003-12-10

    Starting from the previously constructed effective supergravity theory below the scale of U(1) breaking in orbifold compactifications of the weakly coupled heterotic string, we study the effective theory below the scale of supersymmetry breaking by gaugino and matter condensation in a hidden sector. Questions we address include vacuum stability and the masses of the various moduli fields, including those associated with flat directions at the U(1) breaking scale, and of their fermionic superpartners. The issue of soft supersymmetry-breaking masses in the observable sector presents a particularly serious challenge for this class of models.

  11. Approaching Minimal Flavour Violation from an SU(5) × S 4 × U(1) SUSY GUT

    NASA Astrophysics Data System (ADS)

    Dimou, Maria; King, Stephen F.; Luhn, Christoph

    2016-02-01

    We show how approximate Minimal Flavour Violation (MFV) can emerge from an SU(5) Supersymmetric Grand Unified Theory (SUSY GUT) supplemented by an S 4 × U(1) family symmetry, which provides a good description of all quark and lepton (including neutrino) masses, mixings and CP violation. Assuming a SUSY breaking mechanism which respects the family symmetry, we calculate in full explicit detail the low energy mass insertion parameters in the super-CKM basis, including the effects of canonical normalisation and renormalisation group running. We find that the very simple family symmetry S 4 ×U(1) is sufficient to approximately reproduce the effects of low energy MFV.

  12. Dilatometric study of U1-xAmxO2±δ and U1-xCexO2±δ reactive sintering

    NASA Astrophysics Data System (ADS)

    Horlait, Denis; Feledziak, Alex; Lebreton, Florent; Clavier, Nicolas; Prieur, Damien; Dacheux, Nicolas; Delahaye, Thibaud

    2013-10-01

    In order to reduce the radiotoxicity of nuclear fuel waste, the transmutation of americium in U1-xAmxO2±δ dedicated fuels is considered. A convenient route to produce such fuels is reactive sintering from a UO2+δ/AmO2-δ green pellet, i.e., a single heat treatment during which both the densification and the formation of the U1-xAmxO2±δ solid solution occur. The mechanisms of such sintering are however barely known and require experimental data. In this aim, the densification through reactive sintering of a UO2+δ/AmO2-δ sample was monitored by dilatometry. The obtained results were compared to those reported for the formation of the U1-xAmxO2±δ solid solution monitored by in situ high-temperature X-ray diffraction. To assess the use of Ce as a substitute of Am, similar dilatometric studies were also carried out on UO2+δ/CeO2 pellets. Obtained results show that the use of a reactive sintering causes a delay in the densification process associated to the competition between solid solution formation and densification, which yields limitations in pellet final densities. The importance of redox behavior of Am (or Ce) on the achievement of solid solution formation and densification are also discussed, especially based on discrepancies in densification behavior between UO2+δ/AmO2-δ and UO2+δ/CeO2.

  13. 40 CFR Table U-1 to Subpart U of... - CO2 Emission Factors for Common Carbonates

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 22 2013-07-01 2013-07-01 false CO2 Emission Factors for Common Carbonates U Table U-1 to Subpart U of Part 98 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) MANDATORY GREENHOUSE GAS REPORTING Miscellaneous Uses of Carbonate...

  14. 40 CFR Table U-1 to Subpart U of... - CO2 Emission Factors for Common Carbonates

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 22 2012-07-01 2012-07-01 false CO2 Emission Factors for Common Carbonates U Table U-1 to Subpart U of Part 98 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) MANDATORY GREENHOUSE GAS REPORTING Miscellaneous Uses of Carbonate...

  15. Spontaneous SUSY breaking with anomalous U(1) symmetry by meta-stable vacuum

    SciTech Connect

    Nishino, Hiroyuki

    2008-11-23

    We will discuss a SUSY breaking model with anomalous U(1) symmetry. We discard R-symmetry and allow non-renormalizable terms for the model. It will be shown that certain class of models, where the number of positively charged fields is larger than that of negatively charged fields, can have meta-stable SUSY breaking vacuum.

  16. Axionic domain wall number related to U(1)anom global symmetry

    NASA Astrophysics Data System (ADS)

    Kim, Jihn E.

    2016-08-01

    The QCD axion with fa at an intermediate scale, 109 GeV ∼1012 GeV, seems in conflict with the gravity spoil of global symmetries and may face the axionic domain wall problem. We point out that the string compactifications with an anomalous U(1) gauge symmetry, allowing desirable chiral matter spectra, circumvent these two problems simultaneously.

  17. Draft Genome Sequence of Erythromycin- and Oxytetracycline-Sensitive Nocardia seriolae Strain U-1 (NBRC 110359)

    PubMed Central

    Sukeda, Masaki; Shimizu, Masato; Yamane, Jin; Ohnishi, Kouhei; Oshima, Syun-ichirou

    2016-01-01

    In Japan, the emergence of macrolide- and oxytetracycline-resistant strains of Nocardia seriolae has previously been reported. Here, we describe the draft genome sequence of N. seriolae strain U-1, isolated in 2011 from a diseased yellowtail in Kagoshima Prefecture. The draft genome does not have any genes responsible for macrolide and tetracycline resistance. PMID:26798107

  18. 13. DETAIL, U1 JOINT, FROM BELOW AND SOUTH, SHOWING RIVETED ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    13. DETAIL, U1 JOINT, FROM BELOW AND SOUTH, SHOWING RIVETED CONNECTION, AND INTERSECTION OF END POST, VERTICAL, DIAGONAL AND UPPER CHORD MEMBERS AND LATERAL BRACING - Glendale Road Bridge, Spanning Deep Creek Lake on Glendale Road, McHenry, Garrett County, MD

  19. Investigation of gauge-fixed pure U(1) theory at strong coupling

    NASA Astrophysics Data System (ADS)

    Basak, S.; De, Asit K.

    2002-03-01

    We numerically investigate the phase diagram of pure U(1) gauge theory with gauge fixing at strong gauge coupling. The FM-FMD phase transition, which proved useful in defining Abelian lattice chiral gauge theory, persists also at strong gauge coupling. However, there the transition seems no longer to be continuous. At large gauge couplings we find evidences for confinement.

  20. Low scale nonuniversal, nonanomalous U(1)F' in a minimal supersymmetric standard model

    NASA Astrophysics Data System (ADS)

    Chen, Mu-Chun; Huang, Jinrui

    2010-10-01

    We propose a nonuniversal U(1)F' symmetry combined with the minimal supersymmetric standard model. All anomaly cancellation conditions are satisfied without exotic fields other than three right-handed neutrinos. Because our model allows all three generations of chiral superfields to have different U(1)F' charges, upon the breaking of the U(1)F' symmetry at a low scale, realistic masses and mixing angles in both the quark and lepton sectors are obtained. In our model, neutrinos are predicted to be Dirac fermions and their mass ordering is of the inverted hierarchy type. The U(1)F' charges of the chiral superfields also naturally suppress the μ-term and automatically forbid baryon number and lepton number violating operators. While all flavor-changing neutral current constraints in the down quark and charged-lepton sectors can be satisfied, we find that the constraint from D0-D¯0 turns out to be much more stringent than the constraints from the precision electroweak data.

  1. U(1)B-L symmetry restoration and effective neutrino species

    NASA Astrophysics Data System (ADS)

    Ishida, Hiroyuki; Takahashi, Fuminobu

    2014-06-01

    The U(1)B-L symmetry could be restored during inflation, since the BICEP2 results suggest a GUT-scale inflation with the Hubble parameter, Hinf≃1014 GeV, close to the U(1)B-L breaking scale. We consider a scenario in which the B-L Higgs field dominates the Universe after inflation, and mainly decays into the U(1)B-L gauge bosons, whose subsequent decays reheat the Universe. Interestingly, if one of the right-handed neutrinos is extremely light and behaves as dark radiation or hot dark matter, its abundance is determined by the B-L charge assignment and the relativistic degree of freedom in plasma. We find that ΔNeff takes discrete values between 0.188 and 0.220 in the standard model plus three right-handed neutrinos, depending on whether the decay into heavier right-handed neutrinos is kinematically accessible or not. In the fiveness U(1)5 case, we find that ΔNeff takes discrete values between 0.313 and 0.423. The tension between BICEP2 and Planck can be partially relaxed by dark radiation.

  2. Classically conformal U(1 ) ' extended standard model, electroweak vacuum stability, and LHC Run-2 bounds

    NASA Astrophysics Data System (ADS)

    Das, Arindam; Oda, Satsuki; Okada, Nobuchika; Takahashi, Dai-suke

    2016-06-01

    We consider the minimal U(1 ) ' extension of the standard model (SM) with the classically conformal invariance, where an anomaly-free U(1 ) ' gauge symmetry is introduced along with three generations of right-handed neutrinos and a U(1 ) ' Higgs field. Since the classically conformal symmetry forbids all dimensional parameters in the model, the U(1 ) ' gauge symmetry is broken by the Coleman-Weinberg mechanism, generating the mass terms of the U(1 ) ' gauge boson (Z' boson) and the right-handed neutrinos. Through a mixing quartic coupling between the U(1 ) ' Higgs field and the SM Higgs doublet field, the radiative U(1 ) ' gauge symmetry breaking also triggers the breaking of the electroweak symmetry. In this model context, we first investigate the electroweak vacuum instability problem in the SM. Employing the renormalization group equations at the two-loop level and the central values for the world average masses of the top quark (mt=173.34 GeV ) and the Higgs boson (mh=125.09 GeV ), we perform parameter scans to identify the parameter region for resolving the electroweak vacuum instability problem. Next we interpret the recent ATLAS and CMS search limits at the LHC Run-2 for the sequential Z' boson to constrain the parameter region in our model. Combining the constraints from the electroweak vacuum stability and the LHC Run-2 results, we find a bound on the Z' boson mass as mZ'≳3.5 TeV . We also calculate self-energy corrections to the SM Higgs doublet field through the heavy states, the right-handed neutrinos and the Z' boson, and find the naturalness bound as mZ'≲7 TeV , in order to reproduce the right electroweak scale for the fine-tuning level better than 10%. The resultant mass range of 3.5 TeV ≲mZ'≲7 TeV will be explored at the LHC Run-2 in the near future.

  3. Higgs phenomenology in the minimal S U (3 )L×U (1 )X model

    NASA Astrophysics Data System (ADS)

    Okada, Hiroshi; Okada, Nobuchika; Orikasa, Yuta; Yagyu, Kei

    2016-07-01

    We investigate the phenomenology of a model based on the S U (3 )c×S U (3 )L×U (1 )X gauge theory, the so-called 331 model. In particular, we focus on the Higgs sector of the model which is composed of three S U (3 )L triplet Higgs fields and is the minimal form for realizing a phenomenologically acceptable scenario. After the spontaneous symmetry breaking S U (3 )L×U (1 )X→S U (2 )L×U (1 )Y , our Higgs sector effectively becomes that with two S U (2 )L doublet scalar fields, in which the first- and the second-generation quarks couple to a different Higgs doublet from that which couples to the third-generation quarks. This structure causes the flavor-changing neutral current mediated by Higgs bosons at the tree level. By taking an alignment limit of the mass matrix for the C P -even Higgs bosons, which is naturally realized in the case with the breaking scale of S U (3 )L×U (1 )X much larger than that of S U (2 )L×U (1 )Y, we can avoid current constraints from flavor experiments such as the B0-B¯ 0 mixing even for the Higgs bosons masses that are O (100 ) GeV . In this allowed parameter space, we clarify that a characteristic deviation in quark Yukawa couplings of the Standard Model-like Higgs boson is predicted, which has a different pattern from that seen in two Higgs doublet models with a softly broken Z2 symmetry. We also find that the flavor-violating decay modes of the extra Higgs boson, e.g., H /A →t c and H±→t s , can be dominant, and they yield the important signature to distinguish our model from the two Higgs doublet models.

  4. Diphoton excess from hidden U(1) gauge symmetry with large kinetic mixing

    NASA Astrophysics Data System (ADS)

    Takahashi, Fuminobu; Yamada, Masaki; Yokozaki, Norimi

    2016-09-01

    We show that the 750 GeV diphoton excess can be explained by introducing vector-like quarks and hidden fermions charged under a hidden U(1) gauge symmetry, which has a relatively large coupling constant as well as a significant kinetic mixing with U(1)Y. With the large kinetic mixing, the standard model gauge couplings unify around 1017 GeV, suggesting the grand unified theory without too rapid proton decay. Our scenario predicts events with a photon and missing transverse momentum, and its cross section is related to that for the diphoton excess through the kinetic mixing. We also discuss other possible collider signatures and cosmology, including various ways to evade constraints on exotic stable charged particles. In some cases where the 750 GeV diphoton excess is due to diaxion decays, our scenario also predicts triphoton and tetraphoton signals.

  5. Role of the U(1) ghost beyond leading order in a large-Nc expansion

    SciTech Connect

    Hrayr Matevosyan; Anthony Thomas

    2008-09-01

    The 1/Nc expansion is one of the very few methods we have for generating a systematic expansion of QCD at the energy scale relevant to hadron structure. The present formulation of this theory relies on 't Hooft's double-line notation for calculating the leading order of a diagram in the 1/Nc expansion, where the local SU(Nc) gauge symmetry is substituted by a U(Nc) symmetry and the associated U(1) ghost field is ignored. In the current work we demonstrate the insufficiency of this formulation for describing certain non-planar diagrams. We derive a more complete set of Feynman rules that include the U(1) ghost field and provide a useful tool for calculating both color factors and 1/Nc orders of given color-singlet diagrams.

  6. Shaft Sinking at the Nevada Test Site, U1h Shaft Project

    SciTech Connect

    B. Briggs; R. Musick

    2001-03-01

    The U1h Shaft Project is a design/build subcontract to construct one 6.1 meter (m) (20 feet (ft)) finished diameter shaft to a depth of 321.6 m (1,055 ft.) at the Nevada Test Site. Atkinson Construction was subcontracted by Bechtel Nevada to construct the U1h Shaft for the U.S. Department of Energy. The project consists of furnishing and installing the sinking plant, construction of the 321.6 m (1,055 ft.) of concrete lined shaft, development of a shaft station at a depth of 297.5 m (976 ft.), and construction of a loading pocket at the station. The outfitting of the shaft and installation of a new hoist may be incorporated into the project at a later date. This paper will describe the design phase, the excavation and lining operation, shaft station construction and the contractual challenges encountered on this project.

  7. Kagome Chiral Spin Liquid as a Gauged U (1 ) Symmetry Protected Topological Phase

    NASA Astrophysics Data System (ADS)

    He, Yin-Chen; Bhattacharjee, Subhro; Pollmann, Frank; Moessner, R.

    2015-12-01

    While the existence of a chiral spin liquid (CSL) on a class of spin-1 /2 kagome antiferromagnets is by now well established numerically, a controlled theoretical path from the lattice model leading to a low-energy topological field theory is still lacking. This we provide via an explicit construction starting from reformulating a microscopic model for a CSL as a lattice gauge theory and deriving the low-energy form of its continuum limit. A crucial ingredient is the realization that the bosonic spinons of the gauge theory exhibit a U (1 ) symmetry protected topological (SPT) phase, which upon promoting its U (1 ) global symmetry to a local gauge structure ("gauging"), yields the CSL. We suggest that such an explicit lattice-based construction involving gauging of a SPT phase can be applied more generally to understand topological spin liquids.

  8. Instantons and the 5D U(1) gauge theory with extra adjoint

    NASA Astrophysics Data System (ADS)

    Poghossian, Rubik; Samsonyan, Marine

    2009-07-01

    In this paper, we compute the partition function of 5D supersymmetric U(1) gauge theory with extra adjoint matter in general Ω background. It is well known that such partition functions encode very rich topological information. We show in particular that unlike the case with no extra matter, the partition function with extra adjoint at some special values of the parameters directly reproduces the generating function for the Poincare polynomial of the moduli space of instantons. We compare our results with those recently obtained by Iqbal et al (Refined topological vertex, cylindric partitions and the U(1) adjoint theory, arXiv:0803.2260), who used the so-called refined topological vertex method.

  9. Program package for multicanonical simulations of U(1) lattice gauge theory-Second version

    NASA Astrophysics Data System (ADS)

    Bazavov, Alexei; Berg, Bernd A.

    2013-03-01

    A new version STMCMUCA_V1_1 of our program package is available. It eliminates compatibility problems of our Fortran 77 code, originally developed for the g77 compiler, with Fortran 90 and 95 compilers. New version program summaryProgram title: STMC_U1MUCA_v1_1 Catalogue identifier: AEET_v1_1 Licensing provisions: Standard CPC license, http://cpc.cs.qub.ac.uk/licence/licence.html Programming language: Fortran 77 compatible with Fortran 90 and 95 Computers: Any capable of compiling and executing Fortran code Operating systems: Any capable of compiling and executing Fortran code RAM: 10 MB and up depending on lattice size used No. of lines in distributed program, including test data, etc.: 15059 No. of bytes in distributed program, including test data, etc.: 215733 Keywords: Markov chain Monte Carlo, multicanonical, Wang-Landau recursion, Fortran, lattice gauge theory, U(1) gauge group, phase transitions of continuous systems Classification: 11.5 Catalogue identifier of previous version: AEET_v1_0 Journal Reference of previous version: Computer Physics Communications 180 (2009) 2339-2347 Does the new version supersede the previous version?: Yes Nature of problem: Efficient Markov chain Monte Carlo simulation of U(1) lattice gauge theory (or other continuous systems) close to its phase transition. Measurements and analysis of the action per plaquette, the specific heat, Polyakov loops and their structure factors. Solution method: Multicanonical simulations with an initial Wang-Landau recursion to determine suitable weight factors. Reweighting to physical values using logarithmic coding and calculating jackknife error bars. Reasons for the new version: The previous version was developed for the g77 compiler Fortran 77 version. Compiler errors were encountered with Fortran 90 and Fortran 95 compilers (specified below). Summary of revisions: epsilon=one/10**10 is replaced by epsilon/10.0D10 in the parameter statements of the subroutines u1_bmha.f, u1_mucabmha.f, u1wl

  10. First-order phase transition and tricritical point in multiband U (1 ) London superconductors

    NASA Astrophysics Data System (ADS)

    Sellin, Karl A. H.; Babaev, Egor

    2016-02-01

    The order of the superconducting phase transition is a classical problem. Single-component type-2 superconductors exhibit a continuous "inverted-X Y " phase transition, as was first demonstrated for U (1 ) lattice London superconductors by a celebrated duality mapping with subsequent backing by numerical simulations. Here we study this problem in multiband U (1 ) London superconductors and find evidence that by contrast the model has a tricritical point. The superconducting phase transition becomes first order when the Josephson length is sufficiently large compared to the magnetic field penetration length. We present evidence that the fluctuation-induced dipolar interaction between vortex loops makes the phase transition discontinuous. We discuss that this mechanism is also relevant for the phase transitions in multicomponent gauge theories with higher broken symmetry.