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  • 51.
    Eberle, Andrea B.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hessle, Viktoria
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Helbig, Roger
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Dantoft, Widad
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gimber, Niclas
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Splice-Site Mutations Cause Rrp6-Mediated Nuclear Retention of the Unspliced RNAs and Transcriptional Down-Regulation of the Splicing-Defective Genes2010In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 7, p. e11540-Article in journal (Refereed)
    Abstract [en]

    Background: Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes.

    Methodology/Principal Findings: We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster. We have produced S2 cells that express a human β-globin gene with mutated splice sites in intron 2 (mut β-globin). The transcripts encoded by the mut β-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut β-globin transcripts are much lower than those of wild type (wt) ß-globin mRNAs transcribed from the same promoter. We have compared the expression of the mut and wt β-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut β-globin transcripts are processed at the 3′, but the mut β-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut β-globin transcripts are less efficiently released from the transcription site, as shown by FISH, and this defect is restored by depletion of Rrp6 by RNAi. Furthermore, transcription of the mut β-globin gene is significantly impaired as revealed by ChIP experiments that measure the association of the RNA polymerase II with the transcribed genes. We have also shown that the mut β-globin gene shows reduced levels of H3K4me3.

    Conclusions/Significance: Our results show that there are at least two surveillance responses that operate cotranscriptionally in insect cells and probably in all metazoans. One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The other response acts at the transcription level and reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications.

  • 52. Edvardsson, S
    et al.
    Gardner, P
    Poole, A
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hendy, M
    Penny, D
    Moulton, V
    A search for H/ACA snoRNAs in yeast using MFE secondary structure prediction2003In: Bioinformatics, Vol. 19, no 7, p. 865-873Article in journal (Refereed)
  • 53.
    Ekberg, Monica
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Birgander, Pernilla
    Sjöberg, Britt-Marie
    In vivo assay for low-activity mutant forms of Escherichia coli ribonucleotide reductase2003In: Journal of Bacteriology, ISSN 0021-9193, Vol. 185, no 4, p. 1167-1173Article in journal (Refereed)
  • 54.
    Ekberg, Monika
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Birgander, Pernilla
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    In vivo assay for low-activity mutant forms of Escherichia coli ribonucleotide reductase.2003In: J Bacteriol, ISSN 0021-9193, Vol. 185, no 4, p. 1167-73Article in journal (Other academic)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells. In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic Escherichia coli cells. The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR. This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays. E. coli RNR is composed of two homodimeric proteins, R1 and R2. The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site. The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues. Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity. In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.

  • 55.
    Ekdahl, Ylva
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Farahani, Hossein Shahrabi
    Behm, Mikaela
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Lagergren, Jens
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    A-to-I editing of microRNAs in the mammalian brain increases during development2012In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 22, no 8, p. 1477-1487Article in journal (Refereed)
    Abstract [en]

    Adenosine-to-inosine (A-to-I) RNA editing targets double-stranded RNA stem-loop structures in the mammalian brain. It has previously been shown that miRNAs are substrates for A-to-I editing. For the first time, we show that for several definitions of edited miRNA, the level of editing increases with development, thereby indicating a regulatory role for editing during brain maturation. We use high-throughput RNA sequencing to determine editing levels in mature miRNA, from the mouse transcriptome, and compare these with the levels of editing in pri-miRNA. We show that increased editing during development gradually changes the proportions of the two miR-376a isoforms, which previously have been shown to have different targets. Several other miRNAs that also are edited in the seed sequence show an increased level of editing through development. By comparing editing of pri-miRNA with editing and expression of the corresponding mature miRNA, we also show an editing-induced developmental regulation of miRNA expression. Taken together, our results imply that RNA editing influences the miRNA repertoire during brain maturation.

  • 56.
    Engström, Y
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Loseva, O
    Theopold, U
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Proteomics of the Drosophila immune response2004In: Trends in Biotechnology, Vol. 22, no 11, p. 600-605Article in journal (Refereed)
  • 57.
    Ensterö, Mats
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The multi-faceted RNA molecule: Characterization and Function in the regulation of Gene Expression2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In this thesis I have studied the RNA molecule and its function and characteristics in the regulation of gene expression. I have focused on two events that are important for the regulation of the transcriptome: Translational regulation through micro RNAs; and RNA editing through adenosine deaminations.

    Micro RNAs (miRNAs) are ~22 nucleotides long RNA molecules that by semi complementarity bind to untranslated regions of a target messenger RNA (mRNA). The interaction manifests through an RNA/protein complex and act mainly by repressing translation of the target mRNA. I have shown that a pre-cursor miRNA molecule have significantly different information content of sequential composition of the two arms of the pre-cursor hairpin. I have also shown that sequential composition differs between species.

    Selective adenosine to inosine (A-to-I) RNA editing is a post-transcriptional process whereby highly specific adenosines in a (pre-)messenger transcript are deaminated to inosines. The deamination is carried out by the ADAR family of proteins and require a specific sequential and structural landscape for target recognition. Only a handful of messenger substrates have been found to be site selectively edited in mammals. Still, most of these editing events have an impact on neurotransmission in the brain.

    In order to find novel substrates for A-to-I editing, an experimental setup was made to extract RNA targets of the ADAR2 enzyme. In concert with this experimental approach, I have constructed a computational screen to predict specific positions prone to A-to-I editing.

    Further, I have analyzed editing in the mouse brain at four different developmental stages by 454 amplicon sequencing. With high resolution, I present data supporting a general developmental regulation of A-to-I editing. I also present data of coupled editing events on single RNA transcripts suggesting an A-to-I editing mechanism that involve ADAR dimers to act in concert. A different editing pattern is seen for the serotonin receptor 5-ht2c.

  • 58.
    Ensterö, Mats
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Daniel, Chammiran
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Wahlstedt, Helene
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Major, Francois
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Recognition and coupling af A-to-I edited sites are determined by the tertiary structure of the RNA2009In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 20, p. 6916-6926Article in journal (Refereed)
    Abstract [en]

    Adenosine-to-inosine (A-to-I) editing has been shown to be an important mechanism that increases protein diversity in the brain of organisms from human to fly. The family of ADAR enzymes converts some adenosines of RNA duplexes to inosines through hydrolytic deamination. The adenosine recognition mechanism is still largely unknown. Here, to investigate it, we analyzed a set of selectively edited substrates with a cluster of edited sites. We used a large set of individual transcripts sequenced by the 454 sequencing technique. On average, we analyzed 570 single transcripts per edited region at four different developmental stages from embryogenesis to adulthood. To our knowledge, this is the first time, large-scale sequencing has been used to determine synchronous editing events. We demonstrate that edited sites are only coupled within specific distances from each other. Furthermore, our results show that the coupled sites of editing are positioned on the same side of a helix, indicating that the three-dimensional structure is key in ADAR enzyme substrate recognition. Finally, we propose that editing by the ADAR enzymes is initiated by their attraction to one principal site in the substrate.

  • 59.
    Ensterö, Mats
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Daniel, Chammiran
    Wahlstedt, Helene
    Öhman, Marie
    An in-depth survey of A-to-I editing implies a general developmental regulation and coupling of edited sitesManuscript (Other academic)
  • 60.
    Ensterö, Mats
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Åkerborg, Örjan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Wang, Bei
    Furey, Terrence S
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Lagergren, Jens
    A computational screen for site selective A-to-I editing detects novel sites in neuron specific Hu proteins2010In: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 11, no 6Article in journal (Refereed)
    Abstract [en]

    Background

    Several bioinformatic approaches have previously been used to find novel sites of ADAR mediated A-to-I RNA editing in human. These studies have discovered thousands of genes that are hyper-edited in their non-coding intronic regions, especially in alu retrotransposable elements, but very few substrates that are site-selectively edited in coding regions. Known RNA edited substrates suggest, however, that site selective A-to-I editing is particularly important for normal brain development in mammals.

    Results

    We have compiled a screen that enables the identification of new sites of site-selective editing, primarily in coding sequences. To avoid hyper-edited repeat regions, we applied our screen to the alu-free mouse genome. Focusing on the mouse also facilitated better experimental verification. To identify candidate sites of RNA editing, we first performed an explorative screen based on RNA structure and genomic sequence conservation. We further evaluated the results of the explorative screen by determining which transcripts were enriched for A-G mismatches between the genomic template and the expressed sequence since the editing product, inosine (I), is read as guanosine (G) by the translational machinery. For expressed sequences, we only considered coding regions to focus entirely on re-coding events. Lastly, we refined the results from the explorative screen using a novel scoring scheme based on characteristics for known A-to-I edited sites. The extent of editing in the final candidate genes was verified using total RNA from mouse brain and 454 sequencing.

    Conclusions

    Using this method, we identified and confirmed efficient editing at one site in the Gabra3 gene. Editing was also verified at several other novel sites within candidates predicted to be edited. Five of these sites are situated in genes coding for the neuron-specific RNA binding proteins HuB and HuD.

  • 61.
    Esfahani, Shiva Seyedoleslami
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Engström, Ylva
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Activation of an innate immune response in large numbers of permeabilized Drosophila embryos2011In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 35, no 3, p. 263-266Article in journal (Refereed)
    Abstract [en]

    Innate immunity in Drosophila involves the inducible expression and synthesis of antimicrobial peptides. We have previously shown that not only Drosophila larvae and adults, but also embryos, are capable of mounting an immune response after injection of bacterial substances. To simplify genetic dissection of the signaling pathways involved in immune-gene regulation we developed a procedure for permeabilization of large number of embryos and subsequent infiltration with bacterial fragments. This approach, which promoted expression of CecropinA1- and Diptericin-driven β-gal expression in the epidermis of more than 90% of the treated embryos, will enable analysis of mutants that are embryonic lethal. Thus, genes that are involved in essential pleiotrophic functions, in addition to being candidates in immune-regulation will be amenable for analysis of their involvement in the fly's immune defense.

  • 62. Falk, R
    et al.
    Agaton, C
    Kiesler, E
    Jin, S
    Wieslander, L
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Visa, N
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hober, S
    Ståhl, S
    An improval dual expression concept generation high-quality antibodies for proteomics research2003In: Biotechnol. & Appl. Biochem. Immediate PublictionArticle in journal (Refereed)
  • 63.
    Flodin, Veronica S.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The Necessity of Making Visible Concepts with Multiple Meanings in Science Education: The Use of the Gene Concept in a Biology Textbook2009In: Science & Education, ISSN 0926-7220, E-ISSN 1573-1901, Vol. 18, no 1, p. 73-94Article in journal (Refereed)
    Abstract [en]

    The purpose of this study is to analyze variations in how the gene concept is used and conceived in different sub-disciplines in biology. An examination of the development of subject matter and the use of the gene concept in a common college biology textbook shows that the gene concept is far from presented in a consistent way. The study describes and categorizes five different gene concepts used in the textbook; the gene as a trait, an information-structure, an actor, a regulator and a marker. These conceptual differences are not dealt with in an explicit manner. This constitutes one of the sources for confusion when learning about genes and genetics.

  • 64. Fong, Nova
    et al.
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Bentley, David L
    Fast ribozyme cleavage releases transcripts from RNA polymerase II and aborts co-transcriptional pre-mRNA processing2009In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 16, no 9, p. 916-923Article in journal (Refereed)
    Abstract [en]

    Adenosine-to-inosine (A-to-I) editing has been shown to be an important mechanism that increases protein diversity in the brain of organisms from human to fly. The family of ADAR enzymes converts some adenosines of RNA duplexes to inosines through hydrolytic deamination. The adenosine recognition mechanism is still largely unknown. Here, to investigate it, we analyzed a set of selectively edited substrates with a cluster of edited sites. We used a large set of individual transcripts sequenced by the 454 sequencing technique. On average, we analyzed 570 single transcripts per edited region at four different developmental stages from embryogenesis to adulthood. To our knowledge, this is the first time, large-scale sequencing has been used to determine synchronous editing events. We demonstrate that edited sites are only coupled within specific distances from each other. Furthermore, our results show that the coupled sites of editing are positioned on the same side of a helix, indicating that the three-dimensional structure is key in ADAR enzyme substrate recognition. Finally, we propose that editing by the ADAR enzymes is initiated by their attraction to one principal site in the substrate.

  • 65. Freyhult, Eva
    et al.
    Edvardsson, Sverker
    Tamas, Ivaca
    Moulton, Vincent
    Poole, Anthony M
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Fisher: a program for the detection of H/ACA snoRNAs using MFE secondary structure prediction and comparative genomics - assessment and update2008In: BMC Research Notes, Vol. 1, no 49Article, review/survey (Other (popular science, discussion, etc.))
  • 66. Friedrich, Nancy C
    et al.
    Torrents, Eduard
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gibb, Ewan A
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Edgell, David R
    Insertion of a homing endonuclease creates a genes-in-pieces ribonucleotide reductase that retains function2007In: PNAS, Vol. 104, no 15, p. 6176-6181Article in journal (Refereed)
  • 67. Glatz, R
    et al.
    Roberts, H L S
    Li, D
    Sarjan, M
    Theopold, U H
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Asgari, S
    Schmidt, O
    Lectin-induced haemocyte inactivation in insects2004In: Journal of Insect Physiology, Vol. 50, p. 955-963Article in journal (Refereed)
  • 68. Gorodkin, J
    et al.
    Havgaard, J H
    Ensterö, M
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sawera, M
    Jensen, P
    Öhman, M
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Fredholm, M
    MicroRNA sequence motifs reveal asymmetry between the stem arms2006In: Computational Biology and Chemistry, Vol. 30, p. 249-254Article in journal (Refereed)
  • 69. Gorodkin, Jan
    et al.
    Havgaard, Jakob H.
    Ensterö, Mats
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sawera, Milena
    Jensen, Peter
    Öhman, Marie
    Fredholm, Merete
    MicroRNA sequence motifs reveal asymmetry between the stem arms2006In: Computational Biology and Chemistry, ISSN 1476-9271, Vol. 30, no 4, p. 10-Article in journal (Refereed)
  • 70. Gribaldo, Simonetta
    et al.
    Poole, Anthony M.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Daubin, Vincent
    Forterre, Patrick
    Brochier-Armanet, Celine
    The origin of eukaryotes and their relationship with the Archaea: are we at a phylogenomic impasse?2010In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 8, no 10, p. 743-752Article in journal (Refereed)
    Abstract [en]

    The origin of eukaryotes and their evolutionary relationship with the Archaea is a major biological question and the subject of intense debate. In the context of the classical view of the universal tree of life, the Archaea and the Eukarya have a common ancestor, the nature of which remains undetermined. Alternative views propose instead that the Eukarya evolved directly from a bona fide archaeal lineage. Several recent large-scale phylogenomic studies using an array of approaches are divided in supporting either one or the other scenario, despite analysing largely overlapping data sets of universal genes. We examine the reasons for such a lack of consensus and consider how alternative approaches may enable progress in answering this fascinating and as-yet-unresolved question.

  • 71. Gustafsson, Tomas N.
    et al.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Lu, Jun
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Holmgren, Arne
    Bacillus anthracis Thioredoxin Systems, Characterization and Role as Electron Donors for Ribonucleotide Reductase2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 47Article in journal (Refereed)
    Abstract [en]

    Bacillus anthracis is the causative agent of anthrax, which is associated with a high mortality rate. Like several medically important bacteria, B. anthracis lacks glutathione but encodes many genes annotated as thioredoxins, thioredoxin reductases, and glutaredoxin-like proteins. We have cloned, expressed, and characterized three potential thioredoxins, two potential thioredoxin reductases, and three glutaredoxin-like proteins. Of these, thioredoxin 1 (Trx1) and NrdH reduced insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and the manganese-containing type Ib ribonucleotide reductase (RNR) from B. anthracis in the presence of NADPH and thioredoxin reductase 1 (TR1), whereas thioredoxin 2 (Trx2) could only reduce DTNB. Potential TR2 was verified as an FAD-containing protein reducible by dithiothreitol but not by NAD(P)H. The recently discovered monothiol bacillithiol did not work as a reductant for RNR, either directly or via any of the redoxins. The catalytic efficiency of Trx1 was 3 and 20 times higher than that of Trx2 and NrdH, respectively, as substrates for TR1. Additionally, the catalytic efficiency of Trx1 as an electron donor for RNR was 7-fold higher than that of NrdH. In extracts of B. anthracis, Trx1 was responsible for almost all of the disulfide reductase activity, whereas Western blots showed that the level of Trx1 was 15 and 60 times higher than that of Trx2 and NrdH, respectively. Our findings demonstrate that the most important general disulfide reductase system in B. anthracis is TR1/Trx1 and that Trx1 is the physiologically relevant electron donor for RNR. This information may provide a basis for the development of novel antimicrobial therapies targeting this severe pathogen.

  • 72. Hart, K
    et al.
    Nyström, B
    Öhman, M
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Nilsson, L
    Molecular dynamics simulations and free energy calculations of base flipping in dsRNA2005In: RNA, Vol. 11, p. 609-618Article in journal (Refereed)
  • 73. Herwald, Heiko
    et al.
    Theopold, Ulrich
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hemostasis in Invertebrates and Vertebrates: An Evolutionary Excursion2011In: Journal of Innate Immunity, ISSN 1662-811X, E-ISSN 1662-8128, Vol. 3, no 1, p. 1-2Article in journal (Refereed)
  • 74.
    Hessle, Viktoria
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Characterization of RNA exosome in Insect Cells: Role in mRNA Surveillance2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The exosome, an evolutionarily conserved protein complex with exoribonucleolytic activity, is one of the key players in mRNA quality control. Little is known about the functions of the exosome in metazoans. We have studied the role of the exosome in nuclear mRNA surveillance using Chironomus tentans and Drosophila melanogaster as model systems. Studies of the exosome subunits Rrp4 and Rrp6 revealed that both proteins are associated with transcribed genes and nascent pre-mRNPs in C. tentans. We have shown that several exosome subunits interact in vivo with the mRNA-binding protein Hrp59/hnRNP M, and that depleting Hrp59 in D. melanogaster S2 cells by RNAi leads to reduced levels of Rrp4 at the transcription sites. Our results on Rrp4 suggest a model for cotranscriptional quality control in which the exosome is constantly recruited to nascent mRNAs through interactions with specific hnRNP proteins. Moreover, we show that Rrp6 interacts with mRNPs in transit from the gene to the nuclear pore complex, where it is released during early stages of nucleo-cytoplasmic translocation. Furthermore, we show that Rrp6 is enriched in discrete nuclear bodies in the salivary glands of C. tentans and D. melanogaster. In C. tentans, the Rrp6-rich nuclear bodies colocalize with SUMO. We have also constructed D. melanogaster S2 cells expressing human b-globin genes, with either wild type of mutated splice sites, and we have studied the mechanisms by which the cells react to pre-mRNA processing defects. Our results indicate that two surveillance responses operate co-transcriptionally in S2 cells. One requires Rrp6 and retains defective mRNAs at the transcription site. The other one reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications. These observations support the view that multiple mechanisms contribute to co-transcriptional surveillance in insects.

  • 75.
    Hessle, Viktoria
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Björk, Petra
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sokolowski, Marcus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gonzalez de Valdivia, Ernesto
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Silverstein, Rebecca
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Artemenko, Konstantin
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Tyagi, Anu
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Maddalo, Gianluca
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Ilag, Leopold
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Helbig, Roger
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Zubarev, Roman A
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The exosome Associates Cotranscriptionally with the Nascent Pre-mRNP through Interactions with Heterogeneous Nuclear Ribonucleoproteins2009In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 20, no 15, p. 3459-3470Article in journal (Refereed)
    Abstract [en]

    Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality checkpoint takes place cotranscriptionally but little is known about the molecular mechanisms by which the exosome is recruited to the transcribed genes. Here we study the core exosome subunit Rrp4 in two insect model systems, Chironomus and Drosophila. We show that a significant fraction of Rrp4 is associated with the nascent pre-mRNPs and that a specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. Depletion of Hrp59 by RNA interference reduces the levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. Our results lead to a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNP proteins.

  • 76.
    Hessle, Viktoria
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    von Euler, Anne
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    de Valdivia, Ernesto Gonzalez
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Rrp6 is recruited to transcribed genes and accompanies the spliced mRNA to the nuclear pore2012In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 18, no 8, p. 1466-1474Article in journal (Refereed)
    Abstract [en]

    Rrp6 is an exoribonuclease involved in the quality control of mRNA biogenesis. We have analyzed the association of Rrp6 with the Balbiani ring pre-mRNPs of Chironomus tentans to obtain insight into the role of Rrp6 in splicing surveillance. Rrp6 is recruited to transcribed genes and its distribution along the genes does not correlate with the positions of exons and introns. In the nucleoplasm, Rrp6 is bound to both unspliced and spliced transcripts. Rrp6 is released from the mRNPs in the vicinity of the nuclear pore before nucleo-cytoplasmic translocation. We show that Rrp6 is associated with newly synthesized transcripts during all the nuclear steps of gene expression and is associated with the transcripts independently of their splicing status. These observations suggest that the quality control of pre-mRNA splicing is not based on the selective recruitment of the exoribonuclease Rrp6 to unprocessed mRNAs.

  • 77.
    Hoeppner, Marc P.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    White, Simon
    Jeffares, Daniel C.
    Poole, Anthony M.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Evolutionarily Stable Assiciation of Intronic snoRNAs and microRNAs with Their Host Genes2009In: Genome Biology and Evolution, ISSN 1759-6653, Vol. 1, no 1, p. 420-428Article in journal (Refereed)
    Abstract [en]

    Small nucleolar RNAs (snoRNAs) and microRNAs (miRNAs) are integral to a range of processes, including ribosome biogenesis and gene regulation. Some are intron encoded, and this organization may facilitate coordinated coexpression of host gene and RNA. However, snoRNAs and miRNAs are known to be mobile, so intron-RNA associations may not be evolutionarily stable. We have used genome alignments across 11 mammals plus chicken to examine positional orthology of snoRNAs and miRNAs and report that 21% of annotated snoRNAs and 11% of miRNAs are positionally conserved across mammals. Among RNAs traceable to the bird–mammal common ancestor, 98% of snoRNAs and 76% of miRNAs are intronic. Comparison of the most evolutionarily stable mammalian intronic snoRNAs with those positionally conserved among primates reveals that the former are more overrepresented among host genes involved in translation or ribosome biogenesis and are more broadly and highly expressed. This stability is likely attributable to a requirement for overlap between host gene and intronic snoRNA expression profiles, consistent with an ancestral role in ribosome biogenesis. In contrast, whereas miRNA positional conservation is comparable to that observed for snoRNAs, intronic miRNAs show no obvious association with host genes of a particular functional category, and no statistically significant differences in host gene expression are found between those traceable to mammalian or primate ancestors. Our results indicate evolutionarily stable associations of numerous intronic snoRNAs and miRNAs and their host genes, with probable continued diversification of snoRNA function from an ancestral role in ribosome biogenesis.

  • 78.
    Hoeppner, Marc Patrick
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The deep evolutionary roots of non-coding RNA - a comparative genomics approach2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Non-coding RNAs (ncRNA) are a diverse group of genes that do not encode proteins but function exclusively on the level of RNA and were originally suggested to be remnants of a pre-DNA stage of life known as the RNA world. More recent work, however, has uncovered a rich repertoire of previously unknown families with possible consequences for our understanding of the origin and evolution of the modern RNA infrastructure. The main goal of this thesis was therefore to re-examine the evolutionary history of RNAs and theories regarding the transition from an RNA world in light of recent advances in molecular and computational biology.

    Using comparative genomics approaches and sequence data from all domains of life, my work shows that the majority of known RNAs exhibit a highly domain-specific distribution, compatible with an ongoing emergence rather than deep ancestry. Focusing on small nucleolar RNAs (snoRNA), I find that the eukaryote ancestor possessed a complex snoRNA infrastructure, but that intronic snoRNAs are mobile over larger evolutionary time scales. The latter has consequences for predictions made by the Introns-first hypothesis, a framework to explain the emergence of introns in an RNA world and which we revisited in light of advances in our understanding of the evolutionary dynamics of introns.

    A more in-depth analysis of ncRNA mobility across vertebrates found intronic copies of both snoRNAs and miRNAs to be more stable than intergenic ones, suggesting that this arrangement may be a consequence of co-expression. Also, snoRNAs are frequently located in highly expressed genes, in line with their role in ribosome biogenesis. Finally, a closer examination of the genomic distribution of two essential ncRNAs, snoRNA U3 and the spliceosomal RNA U1 shows that both are present in numerous copies across vertebrate genomes. Using next-generation sequencing data, I tested whether this is the result of genetic drift or a requirement for having many copies.

  • 79.
    Hoeppner, Marc Patrick
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gardner, Paul P.
    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus.
    Poole, Anthony M.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Comparative analysis of RNA families reveals distinct repertoires for each domain of lifeManuscript (preprint) (Other academic)
    Abstract [en]

    Some RNAs may date back to an RNA-rich period in the early evolution of life, butmany RNAs are thought to have more recent evolutionary origins. To chart the broadevolutionary history of known RNA families, we performed comparative genomicanalysis of over 3 million RNA annotations spanning 1446 families from the Rfam 10database. We report that 99% of known RNA families are restricted to a singledomain of life, revealing discrete repertoires for each domain. For the 1% of RNAfamilies/clans present in more than one domain, over half show evidence ofhorizontal gene transfer (HGT), and only six RNAs directly trace to the LastUniversal Common Ancestor (LUCA). These results indicate that cellular RNAinfrastructure evolves in a domain-specific manner.

  • 80.
    Hoeppner, Marc Patrick
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Poole, Anthony M.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Comparative Genomics of Eukaryotic Small Nucleolar RNAs Reveals Deep Evolutionary Roots Amidst Ongoing Intragenomic MobilityManuscript (preprint) (Other academic)
    Abstract [en]

    Small nucleolar (sno)RNAs are required for posttranscriptional processing andmodification of ribosomal, spliceosomal and messenger RNAs. There are two broadclasses (C/D and H/ACA), both of which have been characterized in eukaryotes andarchaea. The association with ribosomal RNA processing and modification has led tothe suggestion that snoRNAs are evolutionarily ancient, and date back to the RNAworld. That numerous snoRNAs have been identified in the introns of ribosomalprotein genes has led to alternate views on the origin of this organization. Oneproposal is that intronic snoRNAs predate their surrounding protein-coding exons,the latter being recruited as messenger RNA following the origin of geneticallyencodedprotein synthesis. Another is that intron position reflects selection forcoexpression of snoRNAs and ribosomal components. To gain a clearer insight intothe antiquity of individual snoRNA families and the stability of their genomic location,we examined the evolutionary history of snoRNA families across 44 eukaryotegenomes. Our analysis reveals that dozens of snoRNA families can be traced backto the Last Eukaryotic Common Ancestor (LECA). However, none of the snoRNA1families placed in the LECA are sufficiently similar to characterized archaeal sno-likeRNAs, for us to confidently place specific snoRNA families in the common ancestorof archaea and eukaryotes. In agreement with earlier studies, we can tracenumerous introns to the LECA. However, snoRNAs housed within such positionallyconserved introns are not themselves orthologs. Morevover, our comparativegenomics analysis argues against evolutionarily-stable association betweensnoRNAs and individual host genes — analysis of host gene expression dataindicates that the primary requirement being for hosting intronic snoRNAs is a broadexpression profile. Consistent with mobility over antiquity, we report a case ofdemonstrable intronic snoRNA gain, where an evolutionarily ancient snoRNA hasmigrated into the intron of a mammalian mitochondrial ribosomal protein gene.Together, these data best fit a model wherein snoRNAs are intragenomically mobile,frequently residing in the introns of broadly-expressed protein-coding genes.

  • 81.
    Hoeppner, Marc Patrick
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Poole, Anthony M.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Maintenance of redundant small RNA gene copies over evolutionarytimescales via a retrotransposition motor?Manuscript (preprint) (Other academic)
    Abstract [en]

    We analysed the stability of duplicated, essential RNAs on the backdrop of theirexpression profiles to test whether the data is compatible with functional redundancy ordiversification. Under the former model, the expectation is that copies are equallyexpressed across tissues and subject to high turn-over. The latter model, in contrast,predicts that sub- or neofunctionalization following duplication may lead to a range ofcomplementary expression profiles across tissues. By example of the spliceosomal RNAU1 and snoRNA U3, we find that only few loci are stable over the course of vertebrateevolution and that the majority of copies show little or no expression. We conclude thatthese findings are most compatible with the redundancy model. Interestingly, the deepestloci are associated with a testis-expressed gene, suggesting a possible driving forcebehind the ongoing proliferation that we observe.

  • 82. Hofer, Anders
    et al.
    Crona, Mikael
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Logan, Derek T
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    DNA building blocks: keeping control of manufacture.2012In: Critical reviews in biochemistry and molecular biology, ISSN 1040-9238, E-ISSN 1549-7798, Vol. 47, no 1, p. 50-63Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) is the only source for de novo production of the four deoxyribonucleoside triphosphate (dNTP) building blocks needed for DNA synthesis and repair. It is crucial that these dNTP pools are carefully balanced, since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. In the past few years, the structures of RNRs from several bacteria, yeast and man have been determined in the presence of allosteric effectors and substrates, revealing new information about the mechanisms behind the allosteric regulation. A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme's overall activity by binding ATP (activator) or dATP (inhibitor). The two nucleotides induce formation of different enzyme oligomers, and a recent structure of a dATP-inhibited α(6)β(2) complex from yeast suggested how its subunits interacted non-productively. Interestingly, the oligomers formed and the details of their allosteric regulation differ between eukaryotes and Escherichia coli. Nevertheless, these differences serve a common purpose in an essential enzyme whose allosteric regulation might date back to the era when the molecular mechanisms behind the central dogma evolved.

  • 83.
    Hyrsl, Pavel
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Dobes, Pavel
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Wang, Zhi
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hauling, Thomas
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Wilhelmsson, Christine
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Theopold, Ulrich
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Clotting Factors and Eicosanoids Protect against Nematode Infections2011In: Journal of Innate Immunity, ISSN 1662-811X, Vol. 3, no 1, p. 65-70Article in journal (Refereed)
    Abstract [en]

    We show that hemolymph clotting protects Drosophila melanogaster against infections with an entomopathogenic nematode and its symbiotic bacterium. We also provide biochemical and genetic evidence for an involvement of eicosanoids in the same infection model. Taken together, our results confirm the conserved nature of the immune function of clot formation.

  • 84.
    Högbom, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Huque, Yasmin
    Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Department of Molecular Biology and Functional Genomics.
    Nordlund, Pär
    Department of Molecular Biology and Functional Genomics.
    Crystal structure of the di-iron/radical protein of ribonucleotide reductase from Corynebacterium ammoniagenes.2002In: Biochemistry, ISSN 0006-2960, Vol. 41, no 4, p. 1381-9Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) is the enzyme performing de novo production of the four deoxyribonucleotides needed for DNA synthesis. All mammals as well as some prokaryotes express the class I enzyme which is an alpha(2)beta(2) protein. The smaller of the homodimers, denoted R2, contains a di-iron carboxylate site which, upon reaction with molecular oxygen, generates a stable tyrosyl radical needed for catalysis. The three-dimensional structure of the oxidized class Ib RNR R2 from Corynebacterium ammoniagenes has been determined at 1.85 A resolution and refined to an R-value of 15.8% (R(free) = 21.3%). In addition, structures of both the reduced iron-containing, and manganese-substituted protein have been solved. The C. ammoniagenes R2 has been proposed to be manganese-dependent. The present structure provides evidence that manganese is not oxidized by the protein, in agreement with recent biochemical data, and that no obvious structural abnormalities are seen in the oxidized and reduced iron-containing forms, giving further support that the protein is indeed an iron-dependent RNR R2. The di-manganese structure also provides an explanation for the magnetic properties of this site. The structure of the oxidized C. ammoniagenes R2 also reveals an additional water molecule bridging the radical and the iron site, which has not previously been seen in any other R2 structure and which might have important mechanistic implications.

  • 85.
    Jin, Shao-Bo
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Molecular Cloning and Functional Characterization of Factors Involved in Post-transcriptional Gene Expression2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Gene expression in the eukaryotic cell is a fundamental cellular process, which consists of several distinct steps but extensively coupled to each other. From site of transcription in the nucleus to the cytoplasm, both mRNA and rRNA are associated with a proper set of proteins. These proteins influence RNA processing, transport as well as ribosome maturation. We have tried to take advantage of different model systems to understand the process of eukaryotic gene expression at the post-transcription level. To this end, we have focused on identification and characterization of several specific proteins in the context of mRNP and rRNP particles.

    We have characterized a novel yeast gene MRD1, which encodes a protein with five RNA-binding domains (RBDs) and is essential for viability. Mrd1p is present in the nucleolus and the nucleoplasm. Depletion of Mrd1p leads to a decrease in the synthesis of 18S rRNA and 40S ribosomal subunits. Mrd1p associates with the 35S prerRNA and the U3 snoRNA and is required for the initial processing of pre-rRNA at the A0-A2 sites. The presence of five RBDs in Mrd1p suggests that Mrd1p may function to correctly fold pre-rRNA, a requisite for proper cleavage.

    Meanwhile, an MRD1 homologue, Ct-RBD-1 with six RBDs, has also been identified and shown to involve in ribosome biogenesis in Chironomus tentans. Ct-RBD-1 binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In the cytoplasm, Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that Ct-RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

    We have characterized a novel abundant nucleolar protein, p100 in C. tentans. The p100 protein is located in the fibrillar compartment of the nucleolus, and remains in the nucleolus after digestion with nucleases. This indicates that p100 might be a constituent of the nucleolar proteinaceous framework. Remarkably, p100 is also localized in the brush border in the apical part of the salivary gland cell. These results suggest that it could be involved in coordination of the level of protein production and export from the cell through regulation of the level of rRNA production in the nucleolus.

    We have characterized a Dbp5 homologue in C. tentans, Ct-Dbp5. The protein becomes associated with nascent pre-mRNAs at a large number of active genes, including the Balbiani ring (BR) genes. Ct-Dbp5 is bound to nascent BR pre-mRNP particles and accompanies them through the nucleoplasm and the nuclear pore into the cytoplasm. Nuclear accumulation of Ct-Dbp5 takes place when synthesis and/or export of mRNA are inhibited. Our results indicate that most or all of the shuttling Ct-Dbp5 exiting from the nucleus associated with mRNP. Furthermore, Ct-Dbp5 is present along the mRNP fibril extending into the cytoplasm, supporting the view that Ct-Dbp5 is involved in restructuring the mRNP prior to translation.

    We have shown that the export receptor CRM1 in C. tentans is associated with BR pre-mRNP while transcription takes place. We have also shown that the GTPase Ran binds to BR pre-mRNP, but its binding mainly in the interchromatin. Although both CRM1 and Ran accompany BR pre-mRNP through the nuclear pore, Leptomycin B treatment reveals that a NES-CRM1-RanGTP complex is not essential for export of the BR mRNP. Our results suggest that several export receptors associate with BR mRNP and that these receptors might have redundant functions in the nuclear export of BR mRNP.

    We have analyzed four SR proteins, SC35, ASF/SF2, 9G8 and hrp45, in C. tentans. All four SR proteins genes are expressed in salivary gland cells and in several other tissues in a tissue specific pattern. We found that about 90% of all nascent pre-mRNAs bind all four SR proteins, and that approximately 10% of the pre-mRNAs associate with different subsets of the four SR proteins, suggesting that not all of four SR proteins are needed for processing of pre-mRNA. None of three examined SR proteins leave BR pre-mRNP as splicing is completed. Instead, 9G8 accompanies the mRNP to the cytoplasm, while SC35 and hrp45 leave the BR mRNP at the nuclear side of the nuclear pore complex.

  • 86.
    Jin, Shao-Bo
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Björk, Petra
    Schmekel, Karin
    Ljungdahl, Per O.
    Wieslander, Lars
    Zhao, Jian
    Mrd1p is required for processing of pre-rRNA and for maintenance of steady-state levels of 40S ribosomal subunits in yeast2002In: The Journal of Biological Chemistry, ISSN 1083-351X, no 277, p. 18431-18439Article in journal (Refereed)
  • 87.
    Jin, Shao-Bo
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Zhao, Jian
    Björkroth, Birgitta
    Wieslander, Lars
    Daneholt, Bertil
    The mRNA export factor Dbp5 is associated with Balbiani ring mRNP from the gene to cytoplasm.2002In: The EMBO Journal, ISSN 0261-4189, Vol. 21, no 5, p. 1177-1187Article in journal (Refereed)
  • 88.
    Jin, Shao-Bo
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Zhao, Jian
    Hellman, Ulf
    Wieslander, Lars
    Specific combinations of SR proteins are associated with gene specific nascent pre-mRNAs in vivo.Manuscript (Other academic)
  • 89.
    Jin, Shao-Bo
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Zhao, Jian
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Wieslander, Lars
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    CRM1 and Ran are present but a NES-CRM1-RanGTP complex is not required in Balbiani ring mRNP particles from the gene to the cytoplasm2004In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 117, p. 1553-1566Article in journal (Refereed)
    Abstract [en]

    Messenger RNA is formed from precursors known as pre-mRNA. Theseprecursors associate with proteins to form pre-mRNA-protein(pre-mRNP) complexes. Processing machines cap, splice and polyadenylatethe pre-mRNP and in this way build the mRNP. These processingmachines also affect the export of the mRNP complexes from thenucleus to the cytoplasm. Export to the cytoplasm takes placethrough a structure in the nuclear membrane called the nuclearpore complex (NPC). Export involves adapter proteins in themRNP and receptor proteins that bind to the adapter proteinsand to components of the NPC. We show that the export receptorchromosomal region maintenance protein 1 (CRM1), belonging toa family of proteins known as importin-ß-like proteins,binds to gene-specific Balbiani ring (BR) pre-mRNP while transcriptiontakes place. We also show that the GTPase known as Ran bindsto BR pre-mRNP, and that it binds mainly in the interchromatin.However, we also show using leptomycin B treatment that a NES-CRM1-RanGTPcomplex is not essential for export, even though both CRM1 andRan accompany the BR mRNP through the NPC. Our results thereforesuggest that several export receptors associate with BR mRNPand that these receptors have redundant functions in the nuclearexport of BR mRNP.

  • 90. Johansson, Renzo
    et al.
    Torrents, Eduard
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sprenger, Janina
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    High-resolution crystal structures of the flavoprotein NrdI in oxidized and reduced states: an unusual flavodoxin2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 20, p. 4265-4277Article in journal (Refereed)
    Abstract [en]

    The small flavoprotein NrdI is an essential component of the class Ib ribonucleotide reductase system in many bacteria. NrdI interacts with the class Ib radical generating protein NrdF. It is suggested to be involved in the rescue of inactivated diferric centres or generation of active dimanganese centres in NrdF. Although NrdI bears a superficial resemblance to flavodoxin, its redox properties have been demonstrated to be strikingly different. In particular, NrdI is capable of two-electron reduction, whereas flavodoxins are exclusively one-electron reductants. This has been suggested to depend on a lesser destabilization of the negatively-charged hydroquinone state than in flavodoxins. We have determined the crystal structures of NrdI from Bacillus anthracis, the causative agent of anthrax, in the oxidized and semiquinone forms, at resolutions of 0.96 and 1.4 Å, respectively. These structures, coupled with analysis of all curated NrdI sequences, suggest that NrdI defines a new structural family within the flavodoxin superfamily. The conformational behaviour of NrdI in response to FMN reduction is very similar to that of flavodoxins, involving a peptide flip in a loop near the N5 atom of the flavin ring. However, NrdI is much less negatively charged than flavodoxins, which is expected to affect its redox properties significantly. Indeed, sequence analysis shows a remarkable spread in the predicted isoelectric points of NrdIs, from approximately pH 4-10. The implications of these observations for class Ib ribonucleotide reductase function are discussed.

  • 91.
    Junell, Anna
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Regulation of antimicrobial peptide gene expression in Drosophila melanogaster: Involvement of POU and NF-kB/Rel factors in innate immunity2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The fruit fly, Drosophila melanogaster, has a well-developed immune response, and microbial assault induces a rapid production of potent antimicrobial peptides (AMPs). The aim of this thesis work was to gain deeper knowledge of the regulation of AMPs in Drosophila, by the isolation and characterization of transcription factors involved in AMP gene expression. A yeast screen was designed and used to isolate Drosophila cDNAs coding for novel regulators of the CecropinA1 (CecA1) gene. Three transcription factors belonging to the POU domain (Pdm) family were isolated, Pdm1, Pdm2, and Drifter (Dfr), and subsequently verified as regulators of CecA1 in Drosophila cells. POU proteins are known to regulate a range of developmental processes, but this is the first finding of POU factors controlling AMP gene expression. Dfr and Pdm1 were further analyzed with respect to their in vivo function as AMP gene regulators. Over-expression of Dfr activated several AMP genes in non-infected flies, suggesting that Dfr is involved in constitutive expression of AMP genes. Dfr was shown to bind to a CecA1 upstream enhancer, to which the homeodomain protein Caudal (Cad) previously had been shown to bind. Co-expression of Dfr and Cad promoted very high CecA1 expression, indicating that these two transcription factors act synergistically on CecA1 in tissues where both are expressed. In Pdm1 mutant flies, several AMP genes were highly expressed prior to infection, indicating that Pdm1 functions as a repressor of those genes. However, at least one gene, AttacinA, required Pdm1 for its expression suggesting that Pdm1 has dual functions, acting both as a repressor and activator. Finally, the post-translational activation of the NF-κB/Rel protein Relish in response to infection was investigated in detail. Deletion mapping revealed different functional domains of Relish, and site-directed mutagenesis was used to exactly determine the residues required for endoproteolytic cleavage by a caspase.

  • 92. Junell, Anna
    et al.
    Uvell, Hanna
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Antonsson, Åsa
    Björklund, Gunnel
    Engström, Ylva
    The POU protein Drifter activates antimicrobial peptide gene expression in DrosophilaManuscript (Other academic)
  • 93. Junell, Anna
    et al.
    Uvell, Hanna
    Davis, Monica M
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Edlundh-Rose, Esther
    Antonsson, Åsa
    Pick, Leslie
    Engström, Ylva
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The POU Transcription Factor Drifter/Ventral veinless Regulates Expression of Drosophila Immune Defence Genes2010In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 30, no 14, p. 3672-3684Article in journal (Other academic)
    Abstract [en]

    Innate immunity operates as a first line of defense in multicellular organisms against infections caused by different classes of microorganisms. Antimicrobial peptides (AMPs) are synthesized constitutively in barrier epithelia to protect against microbial attack and are also upregulated in response to infection. Here, we implicate Drifter/Ventral veinless (Dfr/Vvl), a class III POU domain transcription factor, in tissue-specific regulation of the innate immune defense of Drosophila. We show that Dfr/Vvl is highly expressed in a range of immunocompetent tissues, including the male ejaculatory duct, where its presence overlaps with and drives the expression of cecropin, a potent broad-spectrum AMP. Dfr/Vvl overexpression activates transcription of several AMP genes in uninfected flies in a Toll pathway- and Imd pathway-independent manner. Dfr/Vvl activates a CecA1 reporter gene both in vitro and in vivo by binding to an upstream enhancer specific for the male ejaculatory duct. Further, Dfr/Vvl and the homeodomain protein Caudal (Cad) activate transcription synergistically via this enhancer. We propose that the POU protein Dfr/Vvl acts together with other regulators in a combinatorial manner to control constitutive AMP gene expression in a gene-, tissue-, and sex-specific manner, thus promoting a first-line defense against infection in tissues that are readily exposed to pathogens.

  • 94.
    Junell, Anna
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Uvell, Hanna
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Pick, Leslie
    Engström, Ylva
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Isolation of regulators of Drosophila immune defense genes by a double interaction screen in yeast2007In: Insect Biochemistry and Molecular Biology, ISSN 0965-1748, E-ISSN 1879-0240, Vol. 37, no 3, p. 202-212Article in journal (Refereed)
    Abstract [en]

    Innate immunity is a universal and ancient defense system in metazoans against microorganisms. Antimicrobial peptides, which are synthesized both in insects and humans, constitute an endogenous, gene-encoded defense arsenal. In Drosophila, antimicrobial peptides, such as the potent cecropins, are expressed both constitutively in barrier epithelia, as well as systemically in response to infection. Rel/NF-κB proteins are well-known regulators of antimicrobial peptide genes, but very few Rel/NF-κB co-factors and/or tissue-specific regulators have been identified. We performed a double interaction screen in yeast to isolate Drosophila cDNAs coding for direct regulators, as well as Dif co-regulators, of the CecropinA1 gene. Three classes of positive cDNA clones corresponding to 15 Drosophila genes were isolated and further characterized. One of the Dif-independent cDNAs encoded the Rel/NF-κB protein Relish; a well-known activator of antimicrobial peptide genes in Drosophila, demonstrating the applicability of this type of screen for isolating regulators of immune defense. Most interestingly, three transcription factors belonging to the POU domain class of homeodomain proteins, Pdm1, Pdm2 and Dfr/Vvl were isolated as Dif-interacting partners, and subsequently verified as regulators of CecA1 expression in Drosophila cells. The importance of POU proteins in development and differentiation in Drosophila and mammals is well documented, but their role in regulation of Drosophila immune defense genes is a new and essential finding.

  • 95. Kallberg, Yvonne
    et al.
    Segerstolpe, Åsa
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Lackmann, Fredrik
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Persson, Bengt
    Wieslander, Lars
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Evolutionary Conservation of the Ribosomal Biogenesis Factor Rbm19/Mrd1: Implications for Function2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 9, article id e43786Article in journal (Refereed)
    Abstract [en]

    Ribosome biogenesis in eukaryotes requires coordinated folding and assembly of a pre-rRNA into sequential pre-rRNA-protein complexes in which chemical modifications and RNA cleavages occur. These processes require many small nucleolar RNAs (snoRNAs) and proteins. Rbm19/Mrd1 is one such protein that is built from multiple RNA-binding domains (RBDs). We find that Rbm19/Mrd1 with five RBDs is present in all branches of the eukaryotic phylogenetic tree, except in animals and Choanoflagellates, that instead have a version with six RBDs and Microsporidia which have a minimal Rbm19/Mrd1 protein with four RBDs. Rbm19/Mrd1 therefore evolved as a multi-RBD protein very early in eukaryotes. The linkers between the RBDs have conserved properties; they are disordered, except for linker 3, and position the RBDs at conserved relative distances from each other. All but one of the RBDs have conserved properties for RNA-binding and each RBD has a specific consensus sequence and a conserved position in the protein, suggesting a functionally important modular design. The patterns of evolutionary conservation provide information for experimental analyses of the function of Rbm19/Mrd1. In vivo mutational analysis confirmed that a highly conserved loop 5-beta 4-strand in RBD6 is essential for function.

  • 96.
    Karlsson, C
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Korayem, A M
    Scherfer, C
    Loseva, O
    Dushay, M S
    Theopold, U
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Proteomic Analysis of the Drosophila Larval Hemolymph Clot2004In: The Journal of Biological Chemistry, Vol. 279, no 50, p. 52033-52041Article in journal (Refereed)
  • 97.
    Kasrayan, Alex
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Reaction Mechanism and Allosteric Regulation of Class Ia Ribonucleotide Reductase from Escherichia coli2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Ribonculeotide reductase (RNR) plays a key role in catalysing a reaction that provides all living organisms with building blocks for DNA. The RNR enzyme catalyses the conversion of ribonucleotides to their corresponding deoxyribonucleotides, by using free radical chemistry. The class Ia enzyme from Escherichia coli is composed of two components, R1 and R2 proteins. Both components are required for the catalytic reaction. The R2 protein contains a stable tyrosyl free radical, which is directly involved in the catalysis. The R1 protein, contains the active site and two different allosteric sites, which are responsible for regulating the enzyme activity and its substrate specificity.

    An evolutionary conserved active site residue, Asn437, was studied. Employing site directed mutagensis it was shown that the Asn437 is essential for the catalytic reaction. Also using biophysical methods, it was shown that the Asn437 residue participates in the later part of the catalytic reaction.

    By using protein engineering, two conserved overall activity residues His59 and His88 were shown to be involved in the allosteric regulation of enzymatic activity. Furthermore, it was shown by biosensor technique (BIAcore) that the His59 and His88 participate in a communication network that regulates the binding affinity between the R1 and R2 components in response to the presence of certain allosteric effectors.

    The influence of the allosteric effectors and substrate nucleotides on the wild type R1/R2 interaction was investigated using biosensor technique. The presence of allosteric effectors strengthened the affinity between the components R1 and R2 as compared to the affinity in the absence of effectors. Interestingly, when both allosteric effectors and substrate nucleotides were present, the affinity between the R1 and R2 components was noticeably stronger as compared to the situations where only the allosteric effector was present.

    Finally, the residues located in the interaction area of the dimeric R1 protein from E. coli were studied. The aim was to understand their importance for the formation of the active dimeric form of R1. Interestingly, slight alterations of the chemical properties of single side chains resulted in drastic changes in dimer stability, indicating the importance of their interaction ability for dimer formation.

  • 98. Kasrayan, Alex
    et al.
    Larsson Birgander, Pernilla
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Pappalardo, Lucia
    Regnström, Karin
    Westman, MariAnn
    Slaby, Agneta
    Gordon, Euan
    Sjöberg, Britt-Marie
    Enhancement by effectors and substrate nucleotides of R1-R2 interactions in Escherichia coli class Ia ribonucleotide reductase2004In: Journal of Biological chemistry, ISSN 0021-9258, Vol. 279, no 30, p. 31050-31057Article in journal (Refereed)
  • 99.
    Kasrayan, Alex
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Larsson-Birgander, Pernilla
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Pappalardo, Lucia
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Regnström, Karin
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Westman, MariAnn
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Slaby, Agneta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gordon, Euan
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Enhancement by effectors and substrate nucleotides of R1-R2 interactions in Escherichia coli class Ia ribonucleotide reductase.2004In: J Biol Chem, ISSN 0021-9258, Vol. 279, no 30, p. 31050-7Article in journal (Other academic)
    Abstract [en]

    Ribonucleotide reductases are a family of essential enzymes that catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides and provide cells with precursors for DNA synthesis. The different classes of ribonucleotide reductase are distinguished based on quaternary structures and enzyme activation mechanisms, but the components harboring the active site region in each class are evolutionarily related. With a few exceptions, ribonucleotide reductases are allosterically regulated by nucleoside triphosphates (ATP and dNTPs). We have used the surface plasmon resonance technique to study how allosteric effects govern the strength of quaternary interactions in the class Ia ribonucleotide reductase from Escherichia coli, which like all class I enzymes has a tetrameric alpha(2) beta(2) structure. The component alpha(2)called R1 harbors the active site and two types of binding sites for allosteric effector nucleotides, whereas the beta(2) component called R2 harbors the tyrosyl radical necessary for catalysis. Our results show that only the known allosteric effector nucleotides, but not non-interacting nucleotides, promote a specific interaction between R1 and R2. Interestingly, the presence of substrate together with allosteric effector nucleotide strengthens the complex 2-3 times with a similar free energy change as the mutual allosteric effects of substrate and effector nucleotide binding to protein R1 in solution experiments. The dual allosteric effects of dATP as positive allosteric effector at low concentrations and as negative allosteric effector at high concentrations coincided with an almost 100-fold stronger R1-R2 interaction. Based on the experimental setup, we propose that the inhibition of enzyme activity in the E. coli class Ia enzyme occurs in a tight 1:1 complex of R1 and R2. Most intriguingly, we also discovered that thioredoxin, one of the physiological reductants of ribonucleotide reductases, enhances the R1-R2 interaction 4-fold.

  • 100. Kasrayan, Alex
    et al.
    Persson, Annika L
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The Conserved Active Site Asparagine in Class I Ribonucleotide Reductase Is Essential for Catalysis2002In: The Journal of Biological Chemistry, Vol. 277, no 8, p. 5749-5755Article in journal (Refereed)
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