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  • 1.
    Alkemar, Gunnar
    et al.
    Södertörn University, Avdelning Naturvetenskap. Stockholm Universtiy.
    Nygård, Odd
    Södertörn University, Avdelning Naturvetenskap.
    A possible tertiary rRNA interaction between expansion segments ES3 and ES6 in eukaryotic 40S ribosomal subunits2003In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 9, no 1, p. 20-24Article in journal (Refereed)
    Abstract [en]

    Eukaryotic 16S-like ribosomal RNAs contain 12 so-called expansion segments, i.e., sequences not included in the RNA secondary structure core common to eubacteria, archaea, and eukarya. Two of these expansion segments, ES3 and ES6, are juxtaposed in the recent three-dimensional model of the eukaryotic 40S ribosomal subunit. We have analyzed ES3 and ES6 sequences from more than 2900 discrete eukaryotic species, for possible sequence complementarity between the two expansion segments. The data show that ES3 and ES6 could interact by forming a helix consisting of seven to nine contiguous base pairs in almost all analyzed species. We, therefore, suggest that ES3 and ES6 form a direct RNA-RNA contact in the ribosome.

  • 2.
    Alkemar, Gunnar
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Stockholm University.
    Nygård, Odd
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits2004In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 10, no 3, p. 403-411Article in journal (Refereed)
    Abstract [en]

    The 18S rRNA of the small eukaryotic ribosomal subunit contains several expansion segments. Electron microscopy data indicate that two of the largest expansion segments are juxtaposed in intact 40S subunits, and data from phylogenetic sequence comparisons indicate that these two expansion segments contain complementary sequences that could form a direct tertiary interaction on the ribosome. We have investigated the secondary structure of the two expansion segments in the region around the putative tertiary interaction. Ribosomes from yeast, wheat, and mouse-three organisms representing separate eukaryotic kingdoms-were isolated, and the structure of ES3 and part of the ES6 region were analyzed using the single-strand-specific chemical reagents CMCT and DMS and the double-strand-specific ribonuclease V1. The modification patterns were analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The investigated sequences were relatively exposed to chemical and enzymatic modification. This is in line with their indicated location on the surface at the solvent side of the subunit. The complementary ES3 and ES6 sequences were clearly inaccessible to single-strand modification, but available for cleavage by double-strand-specific RNase V1. The results are compatible with a direct helical interaction between bases in ES3 and ES6. Almost identical results were obtained with ribosomes from the three organisms investigated.

  • 3. Andreev, Dmitri
    et al.
    Hauryliuk, Vasili
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Terenin, Ilya
    Dmitriev, Sergey
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Shatsky, Ivan
    The bacterial toxin ReIE induces specific mRNA cleavage in the A site of the eukaryote ribosome2008In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 14, no 2, p. 233-239Article in journal (Refereed)
    Abstract [en]

    ReIE/ReIB is a well-characterized toxin-anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. ReIE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. ReIE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making "ReIE printing" a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site.

  • 4. Avesson, L.
    et al.
    Reimegård, Johan
    Uppsala University.
    Wagner, E. G. H.
    Söderbom, F.
    MicroRNAs in Amoebozoa: Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs2012In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 18, no 10, p. 1771-1782Article in journal (Refereed)
    Abstract [en]

    The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs.

  • 5. Avesson, Lotta
    et al.
    Reimegård, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Wagner, Gerhart Eduard Heinrich
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Söderbom, Fredrik
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    MicroRNAs in Amoebozoa: Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs2012In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 18, no 10, p. 1771-1782Article in journal (Refereed)
    Abstract [en]

    The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a common origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. One example is miRNAs. Their regulation of protein coding genes has been shown to play a vital role in plants and animals but little is known about their role in other organisms. The single cell social amoeba Dictyostelium discoideum could hold the answers to some questions regarding the evolution and function of small RNA pathways. Here we report deep sequencing of small RNAs from three developmental stages of D. discoideum. Analyses of these libraries as well as experimental data reveal the expression of a number of miRNAs, several which have distinct expression patterns during development. We also find miRNAs processed from a hairpin originating from a repetitive element that we believe could represent a pathway for the generation of new miRNAs.

  • 6.
    Babina, Arianne M.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Boston Coll, Dept Biol, Chestnut Hill, MA 02467 USA.
    Parker, Darren J.
    MIT, Dept Biol, 77 Massachusetts Ave, Cambridge, MA 02139 USA.
    Li, Gene-Wei
    MIT, Dept Biol, 77 Massachusetts Ave, Cambridge, MA 02139 USA.
    Meyer, Michelle M.
    Boston Coll, Dept Biol, Chestnut Hill, MA 02467 USA.
    Fitness advantages conferred by the L20-interacting RNA cis-regulator of ribosomal protein synthesis in Bacillus subtilis2018In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 24, no 9, p. 1133-1143Article in journal (Refereed)
    Abstract [en]

    In many bacteria, ribosomal proteins autogenously repress their own expression by interacting with RNA structures typically located in the 5'-UTRs of their mRNA transcripts. This regulation is necessary to maintain a balance between ribosomal proteins and rRNA to ensure proper ribosome production. Despite advances in noncoding RNA discovery and validation of RNA-protein regulatory interactions, the selective pressures that govern the formation and maintenance of such RNA cis-regulators in the context of an organism remain largely undetermined. To examine the impact disruptions to this regulation have on bacterial fitness, we introduced point mutations that abolish ribosomal protein binding and regulation into the RNA structure that controls expression of ribosomal proteins L20 and L35 within the Bacillus subtilis genome. Our studies indicate that removing this regulation results in reduced log phase growth, improper rRNA maturation, and the accumulation of a kinetically trapped or misassembled ribosomal particle at low temperatures, suggesting defects in ribosome synthesis. Such work emphasizes the important role regulatory RNAs play in the stoichiometric production of ribosomal components for proper ribosome composition and overall organism viability and reinforces the potential of targeting ribosomal protein production and ribosome assembly with novel antimicrobials.

  • 7. Babina, Arianne M
    et al.
    Soo, Mark W
    Fu, Yang
    Meyer, Michelle M
    An S6: S18 complex inhibits translation of E. coli rpsF.2015In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 21, no 12, p. 2039-46Article in journal (Refereed)
    Abstract [en]

    More than half of the ribosomal protein operons in Escherichia coli are regulated by structures within the mRNA transcripts that interact with specific ribosomal proteins to inhibit further protein expression. This regulation is accomplished using a variety of mechanisms and the RNA structures responsible for regulation are often not conserved across bacterial phyla. A widely conserved mRNA structure preceding the ribosomal protein operon containing rpsF and rpsR (encoding S6 and S18) was recently identified through comparative genomics. Examples of this RNA from both E. coli and Bacillus subtilis were shown to interact in vitro with an S6:S18 complex. In this work, we demonstrate that in E. coli, this RNA structure regulates gene expression in response to the S6:S18 complex. β-galactosidase activity from a lacZ reporter translationally fused to the 5' UTR and first nine codons of E. coli rpsF is reduced fourfold by overexpression of a genomic fragment encoding both S6 and S18 but not by overexpression of either protein individually. Mutations to the mRNA structure, as well as to the RNA-binding site of S18 and the S6-S18 interaction surfaces of S6 and S18, are sufficient to derepress β-galactosidase activity, indicating that the S6:S18 complex is the biologically active effector. Measurement of transcript levels shows that although reporter levels do not change upon protein overexpression, levels of the native transcript are reduced fourfold, suggesting that the mRNA regulator prevents translation and this effect is amplified on the native transcript by other mechanisms.

  • 8.
    Borg, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Complete kinetic mechanism for recycling of the bacterial ribosome2016In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 22, no 1, p. 10-21Article in journal (Refereed)
    Abstract [en]

    How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of 5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.

  • 9. Bugaeva, Elizaveta Y
    et al.
    Surkov, Serhiy
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Golovin, Andrey V
    Ofverstedt, Lars-Göran
    Skoglund, Ulf
    Isaksson, Leif A
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bogdanov, Alexey A
    Shpanchenko, Olga V
    Dontsova, Olga A
    Structural features of the tmRNA-ribosome interaction2009In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 15, no 12, p. 2312-2320Article in journal (Refereed)
    Abstract [en]

    Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.

  • 10.
    Chavali, Sreenivas
    et al.
    MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
    Bruhn, Sören
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences.
    Tiemann, Katrin
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Sætrom, Pål
    Norwegian University of Science and Technology, Trondheim, Norway.
    Barrenäs, Fredrik
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences.
    Saito, Takaya
    Norwegian University of Science and Technology, Trondheim, Norway.
    Kanduri, Kartiek
    University of Gothenburg, Sweden .
    Wang, Hui
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences.
    Benson, Mikael
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences.
    MicroRNAs act complementarily to regulate disease-related mRNA modules in human diseases2013In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 19, no 11, p. 1552-1562Article in journal (Refereed)
    Abstract [en]

    MicroRNAs (miRNAs) play a key role in regulating mRNA expression, and individual miRNAs have been proposed as diagnostic and therapeutic candidates. The identification of such candidates is complicated by the involvement of multiple miRNAs and mRNAs as well as unknown disease topology of the miRNAs. Here, we investigated if disease-associated miRNAs regulate modules of disease-associated mRNAs, if those miRNAs act complementarily or synergistically, and if single or combinations of miRNAs can be targeted to alter module functions. We first analyzed publicly available miRNA and mRNA expression data for five different diseases. Integrated target prediction and network-based analysis showed that the miRNAs regulated modules of disease-relevant genes. Most of the miRNAs acted complementarily to regulate multiple mRNAs. To functionally test these findings, we repeated the analysis using our own miRNA and mRNA expression data from CD4+ T cells from patients with seasonal allergic rhinitis. This is a good model of complex diseases because of its well-defined phenotype and pathogenesis. Combined computational and functional studies confirmed that miRNAs mainly acted complementarily and that a combination of two complementary miRNAs, miR-223 and miR-139-3p, could be targeted to alter disease-relevant module functions, namely, the release of type 2 helper T-cell (Th2) cytokines. Taken together, our findings indicate that miRNAs act complementarily to regulate modules of disease-related mRNAs and can be targeted to alter disease-relevant functions.

  • 11. Cruz, Jose Almeida
    et al.
    Blanchet, Marc-Frederick
    Boniecki, Michal
    Bujnicki, Janusz M.
    Chen, Shi-Jie
    Cao, Song
    Das, Rhiju
    Ding, Feng
    Dokholyan, Nikolay V.
    Coulbourn Flores, Samuel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Huang, Lili
    Lavender, Christopher A.
    Lisi, Veronique
    Major, Francois
    Mikolajczak, Katarzyna
    Patel, Dinshaw J.
    Philips, Anna
    Puton, Tomasz
    Santalucia, John
    Sijenyi, Fredrick
    Hermann, Thomas
    Rother, Kristian
    Rother, Magdalena
    Serganov, Alexander
    Skorupski, Marcin
    Soltysinski, Tomasz
    Sripakdeevong, Parin
    Tuszynska, Irina
    Weeks, Kevin M.
    Waldsich, Christina
    Wildauer, Michael
    Leontis, Neocles B.
    Westhof, Eric
    RNA-Puzzles: A CASP-like evaluation of RNA three-dimensional structure prediction2012In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 18, no 4, p. 610-625Article in journal (Refereed)
    Abstract [en]

    We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools. We also report the creation of an automated evaluation pipeline to facilitate the analysis of future RNA structure prediction exercises.

  • 12. Dao, E. Han
    et al.
    Poitevin, Frederic
    Sierra, Raymond G.
    Gati, Cornelius
    Rao, Yashas
    Ciftci, Halil Ibrahim
    Aksit, Fulya
    McGurk, Alex
    Obrinski, Trevor
    Mgbam, Paul
    Hayes, Brandon
    de Lichtenberg, Casper
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pardo-Avila, Fatima
    Corsepius, Nicholas
    Zhang, Lindsey
    Seaberg, Matthew H.
    Hunter, Mark S.
    Liang, Mengling
    Koglin, Jason E.
    Wakatsuki, Soichi
    Demirci, Hasan
    Structure of the 30S ribosomal decoding complex at ambient temperature2018In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 24, no 12, p. 1667-1676Article in journal (Refereed)
    Abstract [en]

    The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New light sources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Therm us thermophilus 30S ribosomal subunit decoding complex to 3.45 angstrom resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation to structural studies of RNA and RNA-protein complexes at near-physiological temperatures.

  • 13. Daub, Jennifer
    et al.
    Gardner, Paul P.
    Tate, John
    Ramsköld, Daniel
    KTH.
    Manske, Magnus
    Scott, William G.
    Weinberg, Zasha
    Griffiths-Jones, Sam
    Bateman, Alex
    The RNA WikiProject: Community annotation of RNA families2008In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 14, no 12, p. 2462-2464Article in journal (Refereed)
    Abstract [en]

    The online encyclopedia Wikipedia has become one of the most important online references in the world and has a substantial and growing scientific content. A search of Google with many RNA-related keywords identifies a Wikipedia article as the top hit. We believe that the RNA community has an important and timely opportunity to maximize the content and quality of RNA information in Wikipedia. To this end, we have formed the RNA WikiProject (http://en.wikipedia.org/wiki/Wikipedia: WikiProject_RNA) as part of the larger Molecular and Cellular Biology WikiProject. We have created over 600 new Wikipedia articles describing families of noncoding RNAs based on the Rfam database, and invite the community to update, edit, and correct these articles. The Rfam database now redistributes this Wikipedia content as the primary textual annotation of its RNA families. Users can, therefore, for the first time, directly edit the content of one of the major RNA databases. We believe that this Wikipedia/Rfam link acts as a functioning model for incorporating community annotation into molecular biology databases.

  • 14. Dunham, Christine M.
    et al.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Phelps, Steven S.
    Kelley, Ann C.
    Suzuki, Tsutomu
    Joseph, Simpson
    Ramakrishnan, V.
    Structures of tRNAs with an expanded anticodon loop in the decoding center of the 30S ribosomal subunit2007In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 13, no 6, p. 817-823Article in journal (Refereed)
    Abstract [en]

    During translation, some +1 frameshift mRNA sites are decoded by frameshift suppressor tRNAs that contain an extra base in their anticodon loops. Similarly engineered tRNAs have been used to insert nonnatural amino acids into proteins. Here, we report crystal structures of two anticodon stem–loops (ASLs) from tRNAs known to facilitate +1 frameshifting bound to the 30S ribosomal subunit with their cognate mRNAs. ASLCCCG and ASLACCC (5'–3' nomenclature) form unpredicted anticodon–codon interactions where the anticodon base 34 at the wobble position contacts either the fourth codon base or the third and fourth codon bases. In addition, we report the structure of ASLACGA bound to the 30S ribosomal subunit with its cognate mRNA. The tRNA containing this ASL was previously shown to be unable to facilitate +1 frameshifting in competition with normal tRNAs (Hohsaka et al. 2001), and interestingly, it displays a normal anticodon–codon interaction. These structures show that the expanded anticodon loop of +1 frameshift promoting tRNAs are flexible enough to adopt conformations that allow three bases of the anticodon to span four bases of the mRNA. Therefore it appears that normal triplet pairing is not an absolute constraint of the decoding center.

  • 15. Flores, Samuel
    Turning limited experimental information into 3D models of RNA2010In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 16, no 9, p. 1769-1778Article in journal (Refereed)
  • 16.
    Freihofer, Pietro
    et al.
    Univ Zurich, Inst Med Microbiol, CH-8006 Zurich, Switzerland..
    Akbergenov, Rashid
    Univ Zurich, Inst Med Microbiol, CH-8006 Zurich, Switzerland..
    Teo, Youjin
    Univ Zurich, Inst Med Microbiol, CH-8006 Zurich, Switzerland..
    Juskeviciene, Reda
    Univ Zurich, Inst Med Microbiol, CH-8006 Zurich, Switzerland..
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bottger, Erik C.
    Univ Zurich, Inst Med Microbiol, CH-8006 Zurich, Switzerland..
    Nonmutational compensation of the fitness cost of antibiotic resistance in mycobacteria by overexpression of tlyA rRNA methylase2016In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 22, no 12, p. 1836-1843Article in journal (Refereed)
    Abstract [en]

    Several studies over the last few decades have shown that antibiotic resistance mechanisms frequently confer a fitness cost and that these costs can be genetically ameliorated by intra- or extragenic second-site mutations, often without loss of resistance. Another, much less studied potential mechanism by which the fitness cost of antibiotic resistance could be reduced is via a regulatory response where the deleterious effect of the resistance mechanism is lowered by a physiological alteration that buffers the mutational effect. In mycobacteria, resistance to the clinically used tuberactinomycin antibiotic capreomycin involves loss-of-function mutations in rRNA methylase TIyA or point mutations in 16S rRNA (in particular the A1408G mutation). Both of these alterations result in resistance by reducing drug binding to the ribosome. Here we show that alterations of tlyA gene expression affect both antibiotic drug susceptibility and fitness cost of drug resistance. In particular, we demonstrate that the common resistance mutation A1408G is accompanied by a physiological change that involves increased expression of the tlyA gene. This gene encodes an enzyme that methylates neighboring 16S rRNA position C1409, and as a result of increased TIyA expression the fitness cost of the A1408G mutation is significantly reduced. Our findings suggest that in mycobacteria, a nonmutational mechanism (i.e., gene regulatory) can restore fitness to genetically resistant bacteria. Our results also point to a new and clinically relevant treatment strategy to combat evolution of resistance in multidrug-resistant tuberculosis. Thus, by utilizing antagonistic antibiotic interactions, resistance evolution could be reduced.

  • 17.
    Gonzalez, Grecia M.
    et al.
    Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England..
    Hardwick, Steven W.
    Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England..
    Maslen, Sarah L.
    MRC Lab Mol Biol, Cambridge CB2 0QH, England..
    Skehel, J. Mark
    MRC Lab Mol Biol, Cambridge CB2 0QH, England..
    Holmqvist, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Vogel, Jörg
    Univ Wurzburg, Inst Mol Infect Biol, RNA Biol Grp, D-97080 Wurzburg, Germany.;Univ Wurzburg, Helmholtz Inst RNA Based Infect Res HIRI, D-97080 Wurzburg, Germany..
    Bateman, Alex
    European Bioinformat Inst EMBL EBI, European Mol Biol Lab, Wellcome Genome Campus, Cambridge CB10 1SD, England..
    Luisi, Ben F.
    Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England..
    Broadhurst, R. William
    Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England..
    Structure of the Escherichia coli ProQ RNA-binding protein2017In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 23, no 5, p. 696-711Article in journal (Refereed)
    Abstract [en]

    The protein ProQ has recently been identified as a global small noncoding RNA-binding protein in Salmonella, and a similar role is anticipated for its numerous homologs in divergent bacterial species. We report the solution structure of Escherichia call ProQ, revealing an N-terminal FinO-like domain, a C-terminal domain that unexpectedly has a Tudor domain fold commonly found in eukaryotes, and an elongated bridging intradomain linker that is flexible but nonetheless incompressible. Structure-based sequence analysis suggests that the Tudor domain was acquired through horizontal gene transfer and gene fusion to the ancestral FinO-like domain. Through a combination of biochemical and biophysical approaches, we have mapped putative RNA-binding surfaces on all three domains of ProQ and modeled the protein's conformation in the apo and RNA-bound forms. Taken together, these data suggest how the FinO, Tudor, and linker domains of ProQ cooperate to recognize complex RNA structures and serve to promote RNA-mediated regulation.

  • 18.
    Gustavsson, Marie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ronne, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Evidence that tRNA modifying enzymes are important in vivo targets for 5-fluorouracil in yeast2008In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 14, no 4, p. 666-674Article in journal (Refereed)
    Abstract [en]

    We have screened a collection of haploid yeast knockout strains for increased sensitivity to 5-fluorouracil (5-FU). A total of 138 5-FU sensitive strains were found. Mutants affecting rRNA and tRNA maturation were particularly sensitive to 5-FU, with the tRNA methylation mutant trm10 being the most sensitive mutant. This is intriguing since trm10, like many other tRNA modification mutants, lacks a phenotype under normal conditions. However, double mutants for nonessential tRNA modification enzymes are frequently temperature sensitive, due to destabilization of hypomodified tRNAs. We therefore tested if the sensitivity of our mutants to 5-FU is affected by the temperature. We found that the cytotoxic effect of 5-FU is strongly enhanced at 38 degrees C for tRNA modification mutants. Furthermore, tRNA modification mutants show similar synthetic interactions for temperature sensitivity and sensitivity to 5-FU. A model is proposed for how 5-FU kills these mutants by reducing the number of tRNA modifications, thus destabilizing tRNA. Finally, we found that also wild-type cells are temperature sensitive at higher concentrations of 5-FU. This suggests that tRNA destabilization contributes to 5-FU cytotoxicity in wild-type cells and provides a possible explanation why hyperthermia can enhance the effect of 5-FU in cancer therapy.

  • 19.
    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.

  • 20.
    Huang, Bo
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J.O.
    Byström, Anders S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    An early step in wobble uridine tRNA modification requires the Elongator complex2005In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 11, no 4, p. 424-436Article in journal (Refereed)
    Abstract [en]

    Elongator has been reported to be a histone acetyltransferase complex involved in elongation of RNA polymerase II transcription. In Saccharomyces cerevisiae, mutations in any of the six Elongator protein subunit (ELP1–ELP6) genes or the three killer toxin insensitivity (KTI11–KTI13) genes cause similar pleiotropic phenotypes. By analyzing modified nucleosides in individual tRNA species, we show that the ELP1–ELP6 and KTI11–KTI13 genes are all required for an early step in synthesis of 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) groups present on uridines at the wobble position in tRNA. Transfer RNA immunoprecipitation experiments showed that the Elp1 and Elp3 proteins specifically coprecipitate a tRNA susceptible to formation of an mcm5 side chain, indicating a direct role of Elongator in tRNA modification. The presence of mcm5U, ncm5U, or derivatives thereof at the wobble position is required for accurate and efficient translation, suggesting that the phenotypes of elp1–elp6 and kti11–kti13 mutants could be caused by a translational defect. Accordingly, a deletion of any ELP1–ELP6 or KTI11KTI13 gene prevents an ochre suppressor tRNA that normally contains mcm5U from reading ochre stop codons.

  • 21. Huang, Bo
    et al.
    Lu, Jian
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae.2008In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 14, no 10, p. 2183-2194Article in journal (Refereed)
    Abstract [en]

    We recently showed that the gamma-subunit of Kluyveromyces lactis killer toxin (gamma-toxin) is a tRNA endonuclease that cleaves tRNA(mcm5s2UUC Glu), tRNA(mcm5s2UUU Lys), and tRNA(mcm5s2UUG Gln) 3' of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The 5-methoxycarbonylmethyl (mcm(5)) side chain was important for efficient cleavage by gamma-toxin, and defects in mcm(5) side-chain synthesis correlated with resistance to gamma-toxin. Based on this correlation, a genome-wide screen was performed to identify gene products involved in the formation of the mcm(5) side chain. From a collection of 4826 homozygous diploid Saccharomyces cerevisiae strains, each with one nonessential gene deleted, 63 mutants resistant to Kluyveromyces lactis killer toxin were identified. Among these, eight were earlier identified to have a defect in formation of the mcm(5) side chain. Analysis of the remaining mutants and other known gamma-toxin resistant mutants revealed that sit4, kti14, and KTI5 mutants also have a defect in the formation of mcm(5). A mutant lacking two of the Sit4-associated proteins, Sap185 and Sap190, displays the same modification defect as a sit4-null mutant. Interestingly, several mutants were found to be defective in the synthesis of the 2-thio (s(2)) group of the mcm(5)s(2)U nucleoside. In addition to earlier described mutants, formation of the s(2) group was also abolished in urm1, uba4, and ncs2 mutants and decreased in the yor251c mutant. Like the absence of the mcm(5) side chain, the lack of the s(2) group renders tRNA(mcm5s2UUC Glu) less sensitive to gamma-toxin, reinforcing the importance of the wobble nucleoside mcm(5)s(2)U for tRNA cleavage by gamma-toxin.

  • 22.
    Ieong, Ka-Weng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kwiatkowski, Marek
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids2014In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 20, no 5, p. 632-643Article in journal (Refereed)
  • 23. Ivanova, Natalia
    et al.
    Lindell, Magnus
    Pavlov, Michael
    Holmberg Schiavone, Lovisa
    Södertörn University, School of Life Sciences.
    Wagner, E. Gerhart H.
    Ehrenberg, Mans
    Structure probing of tmRNA in distinct stages of trans-translation2007In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 13, no 5, p. 713-722Article in journal (Refereed)
    Abstract [en]

    Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA ( tmRNA), its helper protein ( small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work we used lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery at different steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reacted with Ala-tmRNAdEF-TudGTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was probed with lead( II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we also analyzed lead( II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. We observed some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNA conformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA is in complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.

  • 24.
    Ivanova, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Lindell, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pavlov, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Holmberg Schiavone, Lovisa
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Structure probing of tmRNA in distinct stages of trans-translation2007In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 13, no 5, p. 713-722Article in journal (Refereed)
    Abstract [en]

    Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helperprotein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work weused lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery atdifferent steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reactedwith Ala-tmRNA EF-Tu GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was                  d      dprobed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we alsoanalyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. Weobserved some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNAconformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA isin complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.

  • 25.
    Johansson, Marcus J O
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Dual function of the tRNA(m(5)U54)methyltransferase in tRNA maturation2002In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 8, no 3, p. 324-335Article in journal (Refereed)
    Abstract [en]

    A 5-methyluridine (m(5)U) residue at position 54 is a conserved feature of bacterial and eukaryotic tRNAs. The methylation of U54 is catalyzed by the tRNA(m5U54)methyltransferase, which in Saccharomyces cerevisiae is encoded by the nonessential TRM2 gene. In this study, we identified four different strains with mutant forms of tRNA(Ser)CGA. The absence of the TRM2 gene in these strains decreased the stability of tRNA(Ser)CGA and induced lethality. Two alleles of TRM2 encoding catalytically inactive tRNA(m5U54)methyltransferases were able to stabilize tRNA(Ser)CGA in one of the mutants, revealing a role for the Trm2 protein per se in tRNA maturation. Other tRNA modification enzymes interacting with tRNA(Ser)CGA in the maturation process, such as Pus4p, Trm1 p, and Trm3p were essential or important for growth of the tRNA(Ser)CGA mutants. Moreover, Lhp1p, a protein binding RNA polymerase III transcripts, was required to stabilize the mutant tRNAs. Based on our results, we suggest that tRNA modification enzymes might have a role in tRNA maturation not necessarily linked to their known catalytic activity.

  • 26.
    Kamel, Wael
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    An Ago2-associated capped transcriptional start site small RNA suppresses adenovirus DNA replication2017In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 23, no 11, p. 1700-1711Article in journal (Refereed)
    Abstract [en]

    Here we show that the adenovirus major late promoter produces a 31-nucleotide transcriptional start site small RNA (MLP-TSS-sRNA) that retains the 7-methylguanosine (m7G)-cap and is incorporated onto Ago2-containing RNA-induced silencing complexes (RISC) in human adenovirus-37 infected cells. RNA polymerase II CLIP (UV-cross linking immunoprecipitation) experiments suggest that the MLP-TSS-sRNA is produced by promoter proximal stalling/termination of RNA polymerase II transcription at the site of the small RNA 3' end. The MLP-TSS-sRNA is highly stable in cells and functionally active, down-regulating complementary targets in a sequence and dose-dependent manner. The MLP-TSS-sRNA is transcribed from the opposite strand to the adenoviral DNA polymerase and preterminal protein mRNAs, two essential viral replication proteins. We show that the MLP-TSS-sRNA act in trans to reduce DNA polymerase and preterminal protein mRNA expression. As a consequence of this, the MLP-TSS-sRNA has an inhibitory effect on the efficiency of viral DNA replication. Collectively, our results suggest that this novel sRNA may serve a regulatory function controlling viral genome replication during a lytic and/or persistent adenovirus infection in its natural host.

  • 27.
    Kang, Wenjing
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Bang-Berthelsen, Claus Heiner
    Holm, Anja
    Houben, Anna J. S.
    Holt Müller, Anne
    Thymann, Thomas
    Pociot, Flemming
    Estivill, Xavier
    Friedländer, Marc R.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Survey of 800+data sets from human tissue and body fluid reveals xenomiRs are likely artifacts2017In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 23, no 4, p. 433-445Article in journal (Refereed)
    Abstract [en]

    miRNAs are small 22-nucleotide RNAs that can post-transcriptionally regulate gene expression. It has been proposed that dietary plant miRNAs can enter the human bloodstream and regulate host transcripts; however, these findings have been widely disputed. We here conduct the first comprehensive meta-study in the field, surveying the presence and abundances of cross-species miRNAs (xenomiRs) in 824 sequencing data sets from various human tissues and body fluids. We find that xenomiRs are commonly present in tissues (17%) and body fluids (69%); however, the abundances are low, comprising 0.001% of host human miRNA counts. Further, we do not detect a significant enrichment of xenomiRs in sequencing data originating from tissues and body fluids that are exposed to dietary intake (such as liver). Likewise, there is no significant depletion of xenomiRs in tissues and body fluids that are relatively separated from the main bloodstream (such as brain and cerebro-spinal fluids). Interestingly, the majority (81%) of body fluid xenomiRs stem from rodents, which are a rare human dietary contribution but common laboratory animals. Body fluid samples from the same studies tend to group together when clustered by xenomiR compositions, suggesting technical batch effects. Last, we performed carefully designed and controlled animal feeding studies, in which we detected no transfer of plant miRNAs into rat blood, or bovine milk sequences into piglet blood. In summary, our comprehensive computational and experimental results indicate that xenomiRs originate from technical artifacts rather than dietary intake.

  • 28. Klaue, Y
    et al.
    Källman, Annika M.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Bonin, M
    Nellen, W
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Biochemical analysis and scanning force microscopy reveal productive and non-productive ADAR2 binding to RNA substrates2003In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 9, no 7, p. 839-846Article in journal (Refereed)
    Abstract [en]

    Scanning force microscopy (SFM) can be used to image biomolecules at high resolution. Here we demonstrate that single-molecule analysis by SFM complements biochemical data on RNA protein binding and can provide information that cannot be obtained by the usual biochemical methods. We have used this method to study the interaction between the RNA editing enzyme ADAR2 and RNA transcripts containing selective and nonselective editing sites. The natural selectively edited R/G site from glutamate receptor subunit B (GluR-B) was inserted into an RNA backbone molecule consisting of a completely double-stranded (ds) central part and incompletely paired ends derived from potato spindle tuber viroid (PSTVd). This molecule was efficiently edited at the R/G site, but promiscuous editing occurred at nonselective sites in the completely double-stranded region. The construct was also used to analyze binding of ADAR2 to wild-type and modified R/G editing sites in relation to binding at other nonselectively edited sites. Editing analysis together with SFM allow us to differentiate between binding and enzymatic activity. ADAR2 has                     been reported to have a general affinity to dsRNA. However, we show that there is a prominent bias for stable binding at sites selectively edited over other edited sites. On the other hand, promiscuous editing at nonselective sites apparently results from transient binding of the enzyme to the substrate. Furthermore, we find distinct sites with nonproductive binding of the enzyme.

  • 29.
    Krzywkowski, Tomasz
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kühnemund, Malte
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection2019In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 25, no 1, p. 82-89Article in journal (Refereed)
    Abstract [en]

    Many approaches exist to detect RNA using complementary oligonucleotides. DNA ligation-based techniques can improve discrimination of subtle sequence variations, but they have been difficult to implement for direct RNA analysis due to the infidelity and inefficiency of most DNA ligases on RNA. In this report, we have systematically studied if ribonucleotide substitutions in padlock probes can provide higher catalytic efficiencies for Chlorella virus DNA ligase (PBCV-1DNA ligase) and T4 RNA ligase 2 (T4Rnl2) on RNA. We provide broad characterization of end-joining fidelity for both enzymes in RNA-templated 3'-OH RNA/5'-pDNA chimeric probe ligation. Both ligases showed increased ligation efficiency toward chimeric substrates on RNA. However, end-joining fidelity of PBCV-1 DNA ligase remained poor, while T4Rnl2 showed a somewhat better end-joining fidelity compared to PBCV-1 DNA ligase. The recently presented invader padlock (iLock) probes overcome the poor end-joining fidelity of PBCV-1 DNA ligase by the requirement of target-dependent 5' flap removal prior to ligation. Here we show that two particular ribonucleotide substitutions greatly improve the activation and ligation rate of chimeric iLock probes on RNA. We characterized the end-joining efficiency and fidelity of PBCV-1 DNA ligase and T4Rnl2 with chimeric iLock probes on RNA and found that both enzymes exhibit high ligation fidelities for single nucleotide poly-morphisms on RNA. Finally, we applied the chimeric probe concept to directly differentiate between human and mouse ACTB mRNA in situ, demonstrating chimeric padlock and iLock probes as superior to their DNA equivalents.

  • 30.
    Kufel, Joanna
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Microbiology.
    Kirsebom, Leif A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Microbiology.
    The P15-loop of Escherichia coli RNase P RNA is an autonomous divalent metal ion binding domain1998In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 4, no 7, p. 777-788Article in journal (Refereed)
    Abstract [en]

    We have studied the structure and divalent metal ion binding of a domain of the ribozyme RNase P RNA that is involved in base pairing with its substrate. Our data suggest that the folding of this internal loop, the P15-loop, is similar irrespective of whether it is part of the full-length ribozyme or part of a model RNA molecule. We also conclude that this element constitutes an autonomous divalent metal ion binding domain of RNase P RNA and our data suggest that certain specific chemical groups within the P15-loop participate in coordination of divalent metal ions. Substitutions of the Sp- and Rp-oxygens with sulfur at a specific position in this loop result in a 2.5-5-fold less active ribozyme, suggesting that Mg2+ binding at this position contributes to function. Our findings strengthen the concept that small RNA building blocks remain basically unchanged when removed from their structural context and thus can be used as models for studies of their potential function and structure within native RNA molecules.

  • 31.
    Kyriakopoulou, Christina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Larsson, Pontus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Liu, Lei
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Schuster, Jens
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Söderbom, Fredrik
    Kirsebom, Leif A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    U1-like snRNAs lacking complementarity to canonical 5' splice sites2006In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 12, no 9, p. 1603-1611Article in journal (Refereed)
    Abstract [en]

    We have detected a surprising heterogeneity among human spliceosomal U1 small nuclear RNA (snRNA). Most interestingly, we have identified three U1 snRNA variants that lack complementarity to the canonical 5' splice site (5'SS) GU dinucleotide. Furthermore, we have observed heterogeneity among the identified variant U1 snRNA genes caused by single nucleotide polymorphism (SNP). The identified snRNAs were ubiquitously expressed in a variety of human tissues representing different stages of development and displayed features of functional spliceosomal snRNAs, i.e., trimethylated cap structures, association with Sm proteins and presence in nuclear RNA-protein complexes. The unanticipated heterogeneity among spliceosomal snRNAs could contribute to the complexity of vertebrates by expanding the coding capacity of their genomes.

  • 32.
    Laxman, Navya
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrinology and mineral metabolism. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Rubin, Carl-Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mallmin, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Nilsson, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Pastinen, Tomi
    Grundberg, Elin
    Kindmark, Andreas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrinology and mineral metabolism. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Global miRNA expression and correlation with mRNA levels in primary human bone cells2015In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 21, no 8, p. 1433-1443Article in journal (Refereed)
    Abstract [en]

    MicroRNAs (miRNAs) are important post-transcriptional regulators that have recently introduced an additional level of intricacy to our understanding of gene regulation. The aim of this study was to investigate miRNA-mRNA interactions that may be relevant for bone metabolism by assessing correlations and interindividual variability in miRNA levels as well as global correlations between miRNA and mRNA levels in a large cohort of primary human osteoblasts (HOBs) obtained during orthopedic surgery in otherwise healthy individuals. We identified differential expression (DE) of 24 miRNAs, and found 9 miRNAs exhibiting DE between males and females. We identified hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b and their target genes as important modulators of bone metabolism. Further, we used an integrated analysis of global miRNA-mRNA correlations, mRNA-expression profiling, DE, bioinformatics analysis, and functional studies to identify novel target genes for miRNAs with the potential to regulate osteoblast differentiation and extracellular matrix production. Functional studies by overexpression and knockdown of miRNAs showed that, the differentially expressed miRNAs hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b target genes highly relevant to bone metabolism, e.g., collagen, type I, alpha 1 (COL1A1), osteonectin (SPARC), Runt-related transcription factor 2 (RUNX2), osteocalcin (BGLAP), and frizzled-related protein (FRZB). These miRNAs orchestrate the activities of key regulators of osteoblast differentiation and extracellular matrix proteins by their convergent action on target genes and pathways to control the skeletal gene expression.

  • 33.
    Lu, Jian
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Esberg, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J O
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Kluyveromyces lactis γ-toxin targets tRNA anticodons.2005In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 11, no 11, p. 1648-1654Article in journal (Refereed)
    Abstract [en]

    Kluyveromyces lactis killer strains secrete a heterotrimeric toxin (zymocin), which causes an irreversible growth arrest of sensitive yeast cells. Despite many efforts, the target(s) of the cytotoxic gamma-subunit of zymocin has remained elusive. Here we show that three tRNA species tRNA(Glu)(mcm(5)s(2)UUC), tRNA(Lys)(mcm(5)s(2)UUU), and tRNA(Gln)(mcm(5)s(2)UUG) are the targets of gamma-toxin. The toxin inhibits growth by cleaving these tRNAs at the 3' side of the modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). Transfer RNA lacking a part of or the entire mcm(5) group is inefficiently cleaved by gamma-toxin, explaining the gamma-toxin resistance of the modification-deficient trm9, elp1-elp6, and kti11-kti13 mutants. The K. lactis gamma-toxin is the first eukaryotic toxin shown to target tRNA.

  • 34.
    Lövgren, Mattias
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Srivastava, Manoj
    Swedish University of Agricultural Sciences, Department of Forest Genetics and Plant Physiology.
    Lundberg, Carina
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Persson, Olof
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wingsle, Gunnar
    Swedish University of Agricultural Sciences, Department of Forest Genetics and Plant Physiology.
    Wikström, Mikael
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The PRC-barrel domain of the ribosome maturation protein RimM mediates binding to ribosomal protein S19 in the 30S ribosomal subunits2004In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 10, no 11, p. 1798-1812Article in journal (Refereed)
    Abstract [en]

    The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes. A DeltarimM mutant is defective in 30S maturation and accumulates 17S rRNA. To study the interaction of RimM with the 30S and its involvement in 30S maturation, RimM amino acid substitution mutants were constructed. A mutant RimM (RimM-YY-->AA), containing alanine substitutions for two adjacent tyrosines within the PRC beta-barrel domain, showed a reduced binding to 30S and an accumulation of 17S rRNA compared to wild-type RimM. The (RimM-YY-->AA) and DeltarimM mutants had significantly lower amounts of polysomes and also reduced levels of 30S relative to 50S compared to a wild-type strain. A mutation in rpsS, which encodes r-protein S19, suppressed the polysome- and 16S rRNA processing deficiencies of the RimM-YY-->AA but not that of the DeltarimM mutant. A mutation in rpsM, which encodes r-protein S13, suppressed the polysome deficiency of both rimM mutants. Suppressor mutations, found in either helices 31 or 33b of 16S rRNA, improved growth of both the RimM-YY-->AA and DeltarimM mutants. However, they suppressed the 16S rRNA processing deficiency of the RimM-YY-->AA mutant more efficiently than that of the DeltarimM mutant. Helices 31 and 33b are known to interact with S13 and S19, respectively, and S13 is known to interact with S19. A GST-RimM but not a GST-RimM(YY-->AA) protein bound strongly to S19 in 30S. Thus, RimM likely facilitates maturation of the region of the head of 30S that contains S13 and S19 as well as helices 31 and 33b.

  • 35.
    Nissan, Tracy
    et al.
    University of Arizona, Tucson, Arizona, USA.
    Parker, Roy
    Computational analysis of miRNA-mediated repression of translation: implications for models of translation initiation inhibition2008In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 14, no 8, p. 1480-1491Article in journal (Refereed)
    Abstract [en]

    The mechanism by which miRNAs inhibit translation has been under scrutiny both in vivo and in vitro. Divergent results have led to the suggestion that miRNAs repress translation by a variety of mechanisms including blocking the function of the cap in stimulating translation. However, these analyses largely only examine the final output of the multistep process of translation. This raises the possibility that when different steps in translation are rate limiting, miRNAs might show different effects on protein production. To examine this possibility, we modeled the process of translation initiation and examined how the effects of miRNAs under different conditions might be explained. Our results suggest that different effects of miRNAs on protein production in separate experiments could be due to differences in rate-limiting steps. This analysis does not rule out that miRNAs directly repress the function of the cap structure, but it demonstrates that the observations used to argue for this effect are open to alternative interpretations. Taking all the data together, our analysis is consistent with the model that miRNAs may primarily repress translation initiation at a late step.

  • 36.
    Nord, Stefan
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bhatt, Monika J
    Baltimore, Maryland 21228, USA.
    Tükenmez, Hasan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Farabaugh, Philip J
    Baltimore, Maryland 21228, USA.
    Wikström, P Mikael
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mutations of ribosomal protein S5 suppress a defect in late-30S ribosomal subunit biogenesis caused by lack of the RbfA biogenesis factor2015In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 21, no 8, p. 1454-1468Article in journal (Refereed)
    Abstract [en]

    The in vivo assembly of ribosomal subunits requires assistance by maturation proteins that are not part of mature ribosomes. One such protein, RbfA, associates with the 30S ribosomal subunits. Loss of RbfA causes cold sensitivity and defects of the 30S subunit biogenesis and its overexpression partially suppresses the dominant cold sensitivity caused by a C23U mutation in the central pseudoknot of 16S rRNA, a structure essential for ribosome function. We have isolated suppressor mutations that restore partially the growth of an RbfA-lacking strain. Most of the strongest suppressor mutations alter one out of three distinct positions in the carboxy-terminal domain of ribosomal protein S5 (S5) in direct contact with helix 1 and helix 2 of the central pseudoknot. Their effect is to increase the translational capacity of the RbfA-lacking strain as evidenced by an increase in polysomes in the suppressed strains. Overexpression of RimP, a protein factor that along with RbfA regulates formation of the ribosome's central pseudoknot, was lethal to the RbfA-lacking strain but not to a wild-type strain and this lethality was suppressed by the alterations in S5. The S5 mutants alter translational fidelity but these changes do not explain consistently their effect on the RbfA-lacking strain. Our genetic results support a role for the region of S5 modified in the suppressors in the formation of the central pseudoknot in 16S rRNA.

  • 37.
    Nordlund, Monica E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, J O Marcus
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    von Pawel-Rammingen, Ulrich
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae.2000In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 6, no 6, p. 844-60Article in journal (Refereed)
    Abstract [en]

    The presence of 5-methyluridine (m5U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U54 nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m5U54)methyltransferase encoding gene trmA+ in E. coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m5U54)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.

  • 38.
    Näsvall, Joakim
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Chen, Peng
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in vivo.2004In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 10, no 10, p. 1662-1673Article in journal (Refereed)
    Abstract [en]

    In Salmonella enterica serovar Typhimurium five of the eight family codon boxes are decoded by a tRNA having the modified nucleoside uridine-5-oxyacetic acid (cmo5U) as a wobble nucleoside present in position 34 of the tRNA. In the proline family codon box, one (tRNAProcmo5UGG) of the three tRNAs that reads the four proline codons has cmo5U34. According to theoretical predictions and several results obtained in vitro, cmo5U34 should base pair with A, G, and U in the third position of the codon but not with C. To analyze the function of cmo5U34 in tRNAProcmo5UGG in vivo, we first identified two genes (cmoA and cmoB) involved in the synthesis of cmo5U34. The null mutation cmoB2 results in tRNA having 5-hydroxyuridine (ho5U34) instead of cmo5U34, whereas the null mutation cmoA1 results in the accumulation of 5-methoxyuridine (mo5U34) and ho5U34 in tRNA. The results suggest that the synthesis of cmo5U34 occurs as follows: U34 -->(?) ho5U -->(CmoB) mo5U -->(CmoA?) cmo5U. We introduced the cmoA1 or the cmoB2 null mutations into a strain that only had tRNAProcmo5UGG and thus lacked the other two proline-specific tRNAs normally present in the cell. From analysis of growth rates of various strains and of the frequency of +1 frameshifting at a CCC-U site we conclude: (1) unexpectedly, tRNAProcmo5UGG is able to read all four proline codons; (2) the presence of ho5U34 instead of cmo5U34 in this tRNA reduces the efficiency with which it reads all four codons; and (3) the fully modified nucleoside is especially important for reading proline codons ending with U or C. Copyright 2004 RNA Society

  • 39.
    Näsvall, S. Joakim
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Chen, Peng
    Björk, Glenn R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Wobble hypothesis revisited: Uridine-5-oxyacetic acid is critical for reading of G-ending codons2007In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 13, no 12, p. 2151-2164Article in journal (Refereed)
    Abstract [en]

    According to Crick's wobble hypothesis, tRNAs with uridine at the wobble position (position 34) recognize A- and G-, but not U- or C-ending codons. However, U in the wobble position is almost always modified, and Salmonella enterica tRNAs containing the modified nucleoside uridine-5-oxyacetic acid (cmo5U34) at this position are predicted to recognize U- (but not C-) ending codons, in addition to A- and G-ending codons. We have constructed a set of S. enterica mutants with only the cmo5U-containing tRNA left to read all four codons in the proline, alanine, valine, and threonine family codon boxes. From the phenotypes of these mutants, we deduce that the proline, alanine, and valine tRNAs containing cmo5U read all four codons including the C-ending codons, while the corresponding threonine tRNA does not. A cmoB mutation, leading to cmo5U deficiency in tRNA, was introduced. Monitoring A-site selection rates in vivo revealed that the presence of cmo5U34 stimulated the reading of CCU and CCC (Pro), GCU (Ala), and GUC (Val) codons. Unexpectedly, cmo5U is critical for efficient decoding of G-ending Pro, Ala, and Val codons. Apparently, whereas G34 pairs with U in mRNA, the reverse pairing (U34-G) requires a modification of U34.

  • 40.
    Obernosterer, G
    et al.
    Austrian Academy of Science.
    Leuschner, PJF
    Austrian Academy of Science.
    Alenius, Matias
    Austrian Academy of Science.
    Martinez, J
    Austrian Academy of Science.
    Post-transcriptional regulation of microRNA expression2006In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 12, no 7, p. 1161-1167Article in journal (Refereed)
    Abstract [en]

    microRNAs ( miRNAs) are endogenous, noncoding similar to 22-nucleotide RNA molecules that have recently emerged as fundamental, post-transcriptional regulators of cognate target gene expression. Many mammalian miRNAs are expressed in a tissue-specific manner, a phenomenon that has so far been attributed to transcriptional regulation. We here show by Northern blots and in situ hybridization experiments that the expression of mammalian miRNAs can be regulated at the post-transcriptional level. In particular, miR-138 is spatially restricted to distinct cell types, while its precursor, pre-miR-138-2, is ubiquitously expressed throughout all tissues analyzed. Furthermore, pre-miR-138-2 is exported from the nucleus to the cytoplasm, suggesting that cleavage of this pre-miRNA by Dicer is restricted to certain tissues and cell types. Thus, differential processing of pre-miRNAs might be an alternative mechanism to control miRNA function.

  • 41.
    Obernosterer, G
    et al.
    Austrian Academy of Science.
    Leuschner, PJF
    Austrian Academy of Science.
    Alenius, Matias
    Austrian Academy of Science.
    Martinez, J
    Austrian Academy of Science.
    Post-transcriptional regulation of microRNA expression2006In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 12, no 7, p. 1161-1167Article in journal (Refereed)
    Abstract [en]

    microRNAs ( miRNAs) are endogenous, noncoding similar to 22-nucleotide RNA molecules that have recently emerged as fundamental, post-transcriptional regulators of cognate target gene expression. Many mammalian miRNAs are expressed in a tissue-specific manner, a phenomenon that has so far been attributed to transcriptional regulation. We here show by Northern blots and in situ hybridization experiments that the expression of mammalian miRNAs can be regulated at the post-transcriptional level. In particular, miR-138 is spatially restricted to distinct cell types, while its precursor, pre-miR-138-2, is ubiquitously expressed throughout all tissues analyzed. Furthermore, pre-miR-138-2 is exported from the nucleus to the cytoplasm, suggesting that cleavage of this pre-miRNA by Dicer is restricted to certain tissues and cell types. Thus, differential processing of pre-miRNAs might be an alternative mechanism to control miRNA function.

  • 42.
    Ren, Yan-Guo
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Martínez, Javier
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Kirsebom, Leif
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Inhibition of Klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides2002In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 8, no 11, p. 1393-1400Article in journal (Refereed)
    Abstract [en]

    Aminoglycosides are known to bind and perturb the function of catalytic RNA. Here we show that they also are potent inhibitors of protein-based catalysis using Escherichia coli Klenow polymerase (pol) and mammalian poly(A)-specific ribonuclease (PARN) as model enzymes. The inhibition was pH dependent and released in a competitive manner by Mg2+. Kinetic analysis showed that neomycin B behaved as a mixed noncompetitive inhibitor. Iron-mediated hydroxyl radical cleavage was used to show that neomycin B interfered with metal-ion binding in the active sites of both enzymes. Our analysis suggests a mechanism of inhibition where the aminoglycoside binds in the active site of the enzyme and thereby displaces catalytically important divalent metal ions. The potential causes of aminoglycoside toxicity and the usage of aminoglycosides to probe, characterize, and perturb metalloenzymes are discussed.

  • 43.
    Repsilber, Dirk
    et al.
    Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
    Wiese, S
    Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
    Rachen, M
    Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
    Schröder, A W
    Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
    Riesner, D
    Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
    Steger, G
    Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
    Formation of metastable RNA structures by sequential folding during transcription: time-resolved structural analysis of potato spindle tuber viroid (-)-stranded RNA by temperature-gradient gel electrophoresis1999In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 5, no 4, p. 574-84Article in journal (Refereed)
    Abstract [en]

    A model of functional elements critical for replication and infectivity of the potato spindle tuber viroid (PSTVd) was proposed earlier: a thermodynamically metastable structure containing a specific hairpin (HP II) in the (-)-strand replication intermediate is essential for template activity during (+)-strand synthesis. We present here a detailed kinetic analysis on how PSTVd (-)-strands fold during synthesis by sequential folding into a variety of metastable structures that rearrange only slowly into the structure distribution of the thermodynamic equilibrium. Synthesis of PSTVd (-)-strands was performed by T7-RNA-polymerase; the rate of synthesis was varied by altering the concentration of nucleoside triphosphates to mimic the in vivo synthesis rate of DNA-dependent RNA polymerase II. With dependence on rate and duration of the synthesis, the structure distributions were analyzed by temperature-gradient gel electrophoresis (TGGE). Metastable structures are generated preferentially at low transcription rates--similar to in vivo rates--or at short transcription times at higher rates. Higher transcription rates or longer transcription times lead to metastable structures in low or undetectable amounts. Instead different structures do gradually appear having a more rod-like shape and higher thermodynamic stability, and the thermodynamically optimal rod-like structure dominates finally. It is concluded that viroids are able to use metastable as well as stable structures for their biological functions.

  • 44.
    Svärd, Staffan G
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Kagardt, Ulrika
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Kirsebom, Leif A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Phylogenetic comparative mutational analysis of the base-pairing between RNase P RNA and its substrate1996In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 2, no 5, p. 463-472Article in journal (Refereed)
    Abstract [en]

    We have studied the base-pairing between the 3'-terminal CCA motif of a tRNA precursor and RNase P RNA by a phylogenetic mutational comparative approach. Thus, various derivatives of the Escherichia coli tRNA(Ser)Su1 precursor harboring all possible substitutions at either the first or the second C of the 3'-terminal CCA motif were generated. Cleavage site selection on these precursors was studied using mutant variants of M1 RNA, the catalytic subunit of E. coli RNase P, carrying changes at positions 292 or 293, which are involved in the interaction with the 3'-terminal CCA motif. From our data we conclude that these two C's in the substrate interact with the well-conserved G292 and G293 through canonical Watson-Crick base-pairing. Cleavage performed using reconstituted holoenzyme complexes suggests that this interaction also occurs in the presence of the C5 protein. Furthermore, we studied the interaction using various derivatives of RNase P RNAs from Mycoplasma hyopneumoniae and Mycobacterium tuberculosis. Our results suggest that the base-pairing between the 3'-terminal CCA motif and RNase P is present also in other bacterial RNase P-substrate complexes and is not limited to a particular bacterial species.

  • 45.
    Tallsjö, Annika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Kufel, Joanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Kirsebom, Leif A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Interaction between Escherichia coli RNase P RNA and the discriminator base results in slow product release1996In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 2, no 4, p. 299-307Article in journal (Refereed)
    Abstract [en]

    We suggested previously that a purine at the discriminator base position in a tRNA precursor interacts with the well-conserved U294 in M1 RNA, the catalytic subunit of Escherichia coli RNase P. Here we investigated this interaction and its influence on the kinetics of cleavage as well as on cleavage site selection. The discriminator base in precursors to tRNA(Tyr)Su3 and tRNA(Phe) was changed from A to C and cleavage kinetics were studied by wild-type M1 RNA and a mutant M1 RNA carrying the compensatory substitution of a U to a G at position 294 in M1 RNA. Our data suggest that the discriminator base interacts with the residue at position 294 in M1 RNA. Although product release is a rate-limiting step both in the absence and in the presence of this interaction, its presence results in a significant reduction in the rate of product release. In addition, we studied cleavage site selection on various tRNA(His) precursor derivatives. These precursors carry a C at the discriminator base position. The results showed that the mutant M1 RNA harboring a G at position 294 miscleaved a wild-type tRNA(His) precursor and a tRNA(His) precursor carrying an inosine at the cleavage site. The combined data suggest a functional interaction between the discriminator base and the well-conserved U294 in M1 RNA. This interaction is suggested to play an important role in determining the rate of product release during multiple turnover cleavage of tRNA precursors by M1 RNA as well as in cleavage site selection.

  • 46. van der Horst, Sjors
    et al.
    Snel, Berend
    Hanson, Johannes
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Molecular Plant Physiology, Institute of Environmental Biology, Utrecht University, Utrecht, The Netherlands; Theoretical Biology and Bioinformatics, Department of Biology, Utrecht University, Utrecht, The Netherlands .
    Smeekens, Sjef
    Novel pipeline identifies new upstream ORFs and non-AUG initiating main ORFs with conserved amino acid sequences in the 5 ' leader of mRNAs in Arabidopsis thaliana2019In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 25, no 3, p. 292-304Article in journal (Refereed)
    Abstract [en]

    Eukaryotic mRNAs contain a 5' leader sequence preceding the main open reading frame (mORF) and, depending on the species, 20%-50% of eukaryotic mRNAs harbor an upstream ORF (uORF) in the 5' leader. An unknown fraction of these uORFs encode sequence conserved peptides (conserved peptide uORFs, CPuORFs). Experimentally validated CPuORFs demonstrated to regulate the translation of downstream mORFs often do so in a metabolite concentration-dependent manner. Previous research has shown that most CPuORFs possess a start codon context suboptimal for translation initiation, which turns out to be favorable for translational regulation. The suboptimal initiation context may even include non-AUG start codons, which makes CPuORFs hard to predict. For this reason, we developed a novel pipeline to identify CPuORFs unbiased of start codon using well-annotated sequence data from 31 eudicot plant species and rice. Our new pipeline was able to identify 29 novel Arabidopsis thaliana (Arabidopsis) CPuORFs, conserved across a wide variety of eudicot species of which 15 do not initiate with an AUG start codon. In addition to CPuORFs, the pipeline was able to find 14 conserved coding regions directly upstream and in frame with the mORF, which likely initiate translation on a non-AUG start codon. Altogether, our pipeline identified highly conserved coding regions in the 5' leaders of Arabidopsis transcripts, including in genes with proven functional importance such as LHY, a key regulator of the circadian clock, and the RAPTOR1 subunit of the target of rapamycin (TOR) kinase.

  • 47.
    Zhang, Jingji
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ieong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mellenius, Harriet
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Proofreading neutralizes potential error hotspots in genetic code translation by transfer RNAs2016In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 22, no 6, p. 896-904Article in journal (Refereed)
    Abstract [en]

    The ribosome uses initial and proofreading selection of aminoacyl-tRNAs for accurate protein synthesis. Anomalously high initial misreading in vitro of near-cognate codons by tRNAHis and tRNAGlu suggested potential error hotspots in protein synthesis, but in vivo data suggested their partial neutralization. To clarify the role of proofreading in this error reduction, we varied the Mg2+ ion concentration to calibrate the total accuracy of our cell-free system to that in the living Escherichia coli cell. We found the total accuracy of tRNA selection in our system to vary by five orders of magnitude depending on tRNA identity, type of mismatch, and mismatched codon position. Proofreading and initial selection were positively correlated at high, but uncorrelated at low initial selection, suggesting hyperactivated proofreading as a means to neutralize potentially disastrous initial selection errors.

  • 48.
    Zhao, Yani
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Holmgren, Benjamin T.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hinas, Andrea
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    The conserved SNARE SEC-22 localizes to late endosomes and negatively regulates RNA interference in Caenorhabditis elegans2017In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 23, no 3, p. 297-307Article in journal (Refereed)
    Abstract [en]

    Small RNA pathways, including RNA interference (RNAi), play crucial roles in regulation of gene expression. Initially considered to be cytoplasmic, these processes have later been demonstrated to associate with membranes. For example, maturation of late endosomes/multivesicular bodies (MVBs) is required for efficient RNAi, whereas fusion of MVBs to lysosomes appears to reduce silencing efficiency. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane fusion and are thus at the core of membrane trafficking. In spite of this, no SNARE has previously been reported to affect RNAi. Here, we demonstrate that in Caenorhabditis elegans, loss of the conserved SNARE SEC-22 results in enhanced RNAi upon ingestion of double-stranded RNA. Furthermore, SEC-22 overexpression inhibits RNAi in wild-type animals. We find that overexpression of SEC-22 in the target tissue (body wall muscle) strongly suppresses the sec-22(-) enhanced RNAi phenotype, supporting a primary role for SEC-22 in import of RNAi silencing signals or cell autonomous RNAi. A functional mCherry:: SEC-22 protein localizes primarily to late endosomes/MVBs and these compartments are enlarged in animals lacking sec-22. SEC-22 interacts with late endosome-associated RNA transport protein SID-5 in a yeast two-hybrid assay and functions in a sid-5-dependent manner. Taken together, our data indicate that SEC-22 reduces RNAi efficiency by affecting late endosome/MVB function, for example, by promoting fusion between late endosomes/MVBs and lysosomes. To our knowledge, this is the first report of a SNARE with a function in small RNA-mediated gene silencing.

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