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  • 1. Anjard, Christophe
    et al.
    Söderbom, Fredrik
    Dept. of Microbiology, Swedish University of Agricultural Sciences Box 7025, 750 07 Uppsala, Sweden .
    Loomis, William F
    Requirements for the adenylyl cyclases in the development of Dictyostelium2001In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 128, no 18, p. 3649-3654Article in journal (Refereed)
    Abstract [en]

    It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA- mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA- PKA-C(over) strain. When developed at high cell densities, acrA- acaA- PKA-C(over) cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high.

  • 2. 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.

  • 3. Boesler, Carsten
    et al.
    Kruse, Janis
    Söderbom, Fredrik
    Department of Molecular Biology, Uppsala Biomedical Center, Swedish University of Agricultural Sciences, S-75124 Uppsala, Sweden .
    Hammann, Christian
    Sequence and generation of mature ribosomal RNA transcripts in Dictyostelium discoideum2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 20, p. 17693-17703Article in journal (Refereed)
    Abstract [en]

    The amoeba Dictyostelium discoideum is a well established model organism for studying numerous aspects of cellular and developmental functions. Its ribosomal RNA (rRNA) is encoded in an extrachromosomal palindrome that exists in ∼100 copies in the cell. In this study, we have set out to investigate the sequence of the expressed rRNA. For this, we have ligated the rRNA ends and performed RT-PCR on these circular RNAs. Sequencing revealed that the mature 26 S, 17 S, 5.8 S, and 5 S rRNAs have sizes of 3741, 1871, 162, and 112 nucleotides, respectively. Unlike the published data, all mature rRNAs of the same type uniformly display the same start and end nucleotides in the analyzed AX2 strain. We show the existence of a short lived primary transcript covering the rRNA transcription unit of 17 S, 5.8 S, and 26 S rRNA. Northern blots and RT-PCR reveal that from this primary transcript two precursor molecules of the 17 S and two precursors of the 26 S rRNA are generated. We have also determined the sequences of these precursor molecules, and based on these data, we propose a model for the maturation of the rRNAs in Dictyostelium discoideum that we compare with the processing of the rRNA transcription unit of Saccharomyces cerevisiae.

  • 4. Crona, Mikael
    et al.
    Avesson, Lotta
    Sahlin, Margareta
    Lundin, Daniel
    Hinas, Andrea
    Klose, Ralph
    Söderbom, Fredrik
    Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, SE-75124 Uppsala, Sweden .
    Sjöberg, Britt-Marie
    A Rare Combination of Ribonucleotide Reductases in the Social Amoeba Dictyostelium discoideum2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 12, p. 8198-208Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) catalyze the only pathway for de novo synthesis of deoxyribonucleotides needed for DNA replication and repair. The vast majority of eukaryotes encodes only a class I RNR, but interestingly some eukaryotes, including the social amoeba Dictyostelium discoideum, encode both a class I and a class II RNR. The amino acid sequence of the D. discoideum class I RNR is similar to other eukaryotic RNRs, whereas that of its class II RNR is most similar to the monomeric class II RNRs found in Lactobacillus spp. and a few other bacteria. Here we report the first study of RNRs in a eukaryotic organism that encodes class I and class II RNRs. Both classes of RNR genes were expressed in D. discoideum cells, although the class I transcripts were more abundant and strongly enriched during mid-development compared with the class II transcript. The quaternary structure, allosteric regulation, and properties of the diiron-oxo/radical cofactor of D. discoideum class I RNR are similar to those of the mammalian RNRs. Inhibition of D. discoideum class I RNR by hydroxyurea resulted in a 90% reduction in spore formation and decreased the germination viability of the surviving spores by 75%. Class II RNR could not compensate for class I inhibition during development, and an excess of vitamin B12 coenzyme, which is essential for class II activity, did not improve spore formation. We suggest that class I is the principal RNR during D. discoideum development and growth and is important for spore formation, possibly by providing dNTPs for mitochondrial replication.

  • 5. He, Lin
    et al.
    Söderbom, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, Gerhart
    Binnie, Uta
    Binns, Nigel
    Masters, Millicent
    PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of ColE1-related plasmids1993In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 9, no 6, p. 1131-1142Article in journal (Refereed)
    Abstract [en]

    The replication of ColE1-related plasmids is controlled by an unstable antisense RNA, RNAI, which can interfere with the successful processing of the RNAII primer of replication. We show here that a host protein, PcnB, supports replication by promoting the decay of RNAI. In bacterial strains deleted for PcnB a stable, active form of RNAI, RNAI*, which appears to be identical to the product of 5'-end processing by RNAase E, accumulates. This leads to a reduction in plasmid copy number. We show, using a GST-PcnB fusion protein, that PcnB does not interfere with RNAI/RNAII binding in vitro. The fusion protein, like PcnB, has polyadenylating activity and is able to polyadenylate RNAI (and also another antisense RNA, CopA) in vitro.

  • 6.
    Hällman, Jimmie
    et al.
    KTH Royal Institute of Technology.
    Avesson, Lotta
    Reimegård, Johan
    Käller, Max
    Söderbom, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Identification and verification of microRNAs by high-throughput sequencing2013In: Dictyostelium discoideum Protocols / [ed] Ludwig Eichinger and Francisco Rivero, Humana Press, 2013, 2, Vol. 983, p. 125-138Chapter in book (Other academic)
    Abstract [en]

    High-throughput sequencing methods have become invaluable for detection and analysis of small RNAs. The results are millions of sequences that need to be carefully analyzed by computational methods and preferentially verified by different experimental techniques. Here we describe how to use high-throughput sequencing followed by bioinformatics and northern blot to identify one particular class of small RNA, microRNAs.

  • 7.
    Liao, Zhen
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Kjellin, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Dahlén, Helena
    Söderbom, Fredrik
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    The Dictyostelium discoideum Argonaute protein AgnE regulates cell growthManuscript (preprint) (Other academic)
    Abstract [en]

    Argonaute proteins play essential roles in the RNA interference (RNAi) pathways in eukaryotes. The members of this conserved class of proteins are guided to their target RNAs by their associated small RNAs and can thereby regulate gene expression. The number and function of Argonautes varies depending on the organism but it is clear that they together with their interacting small RNAs constitute the core of the RNA induced silencing complex, RISC. Little is known about the Argonautes in the social amoeba Dictyostelium discoideum, a unicellular organism that can go through multicellular development and evolutionary is placed between plants and animals. In this study, we investigated the phenotypic consequences of deleting the genes for three Argonautes, AgnB, AgnC, and AgnE in D. discoideum. All three Argonautes have an effect on growth since depletion of AgnB and AgnC impaired growth while AgnE depletion, surprisingly, resulted in faster cell division. The intriguing role of AgnE in growth regulation prompted us to further study this protein. We expressed an AgnE-GFP fusion protein and showed that this localize in the cytoplasm. High-throughput sequencing of mRNA from growing agnE- and wt cells showed that genes required for the nucleobase biosynthetic process as well as genes for ribosomal proteins are upregulated in the agnE knock-out strain, which is in line with its faster growth rate. Furthermore, the RNAi related gene encoding RNA-dependent RNA polymerase C is downregulated, which may result in accumulation of miRNAs. The possible connection between growth rate and miRNA levels was explored by analyzing growth rate in a strain depleted of miRNAs, i.e. where the gene for the Dicer-like protein DrnB had been knocked out. This strain grew much slower than wt and this phenotype could not be rescued by disrupting agnE in the drnB- background. This suggests that DrnB acts upstream of AgnE in regulating D. discoideum growth.

  • 8. Liao, Zhen
    et al.
    Kjellin, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Fransson, Åsa
    Söderbom, Fredrik
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Functional analyses of RNA interference effectors in Dictyostelium discoideum during growth and developmentManuscript (preprint) (Other academic)
    Abstract [en]

    RNA interference (RNAi) is a widespread biological process, which regulates gene expression in eukaryotic cells. A complex of proteins and small RNAs, RISC, mediates this gene regulation. Central to the function of RISC are the Argonaute effector proteins, which bind the small RNAs, e.g. micro (mi)RNAs and small interfering (si)RNAs. It has previously been shown that RNAi is important to control transposon mobilization in the social amoeba Dictyostelium discoideum, a unicellular eukaryote that upon starvation enters a multicellular developmental program. Information concerning the five Argonautes in D. discoideum is scarce but several of them appear to inhibit transposon mobilization by RNAi related mechanisms. In a recent study, we showed that three of the Argonautes in D. discoideum are involved in controlling cell division. In this study, we perform mRNA- and small RNA-seq. from growing and developing cells, combined with phenotypic studies of D. discoideum strains depleted of AgnB, AgnC, and AgnE. The previously observed effect on cell division, i.e. faster growth for agnE-, and slower for agnB- and agnC- cells, is associated with increased and decreased expression, respectively, of genes involved in nucleotide metabolism. Furthermore, all three argonautes appear to be involved in downregulation of ribosomal protein genes during development while AgnE also contributes to reduced expression of protein coding genes during growth. These effects are likely mediated by small RNAs. We further report the subcellular localization of the three Argonautes, where AgnB is mainly localized in the cytoplasm, AgnC in the nucleus, and the previously reported cytoplasmic localization for AgnE was confirmed. Finally, we present data indicating that AgnB is interacting with miRNAs, suggesting that this Argonaute is involved in miRNA mediated gene regulation in D. discoideum.  Taken together, our data indicate that none of the Argonautes components are essential for cell division and development, but all participate in fine-tuning of gene expression for optimal growth and synchronous multicellular development.

  • 9.
    Liao, Zhen
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Kjellin, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Höppner, Marc P.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Christian-Albrechts-University of Kiel, Institute of Clinical Molecular Biology, Kiel, Germany.
    Grabherr, Manfred
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Söderbom, Fredrik
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum2018In: RNA Biology, ISSN 1547-6286Article in journal (Refereed)
    Abstract [en]

    Micro (mi)RNAs regulate gene expression in many eukaryotic organisms where they control diverse biological processes. Their biogenesis, from primary transcripts to mature miRNAs, have been extensively characterized in animals and plants, showing distinct differences between these phylogenetically distant groups of organisms. However, comparably little is known about miRNA biogenesis in organisms whose evolutionary position is placed in between plants and animals and/or in unicellular organisms. Here, we investigate miRNA maturation in the unicellular amoeba Dictyostelium discoideum, belonging to Amoebozoa, which branched out after plants but before animals. High-throughput sequencing of small RNAs and poly(A)-selected RNAs demonstrated that the Dicer-like protein DrnB is required, and essentially specific, for global miRNA maturation in D. discoideum. Our RNA-seq data also showed that longer miRNA transcripts, generally preceded by a T-rich putative promoter motif, accumulate in a drnB knock-out strain. For two model miRNAs we defined the transcriptional start sites (TSSs) of primary (pri)-miRNAs and showed that they carry the RNA polymerase II specific m7G-cap. The generation of the 3’-ends of these pri-miRNAs differs, with pri-mir-1177 reading into the downstream gene, and pri-mir-1176 displaying a distinct end. This 3´-end is processed to shorter intermediates, stabilized in DrnB-depleted cells, of which some carry a short oligo(A)-tail. Furthermore, we identified 10 new miRNAs, all DrnB dependent and developmentally regulated. Thus, the miRNA machinery in D. discoideum shares features with both plants and animals, which is in agreement with its evolutionary position and perhaps also an adaptation to its complex lifestyle: unicellular growth and multicellular development.

  • 10. Masson, Patrick
    et al.
    Lundin, Daniel
    Söderbom, Fredrik
    Department of Molecular Biology, Swedish University of Agricultural Sciences (SLU), Uppsala Biomedical Center (BMC), Box 590, S-75124 Uppsala, Sweden .
    Young, Patrick
    Characterization of a REG/PA28 proteasome activator homolog in Dictyostelium discoideum indicates that the ubiquitin- and ATP-independent REGgamma proteasome is an ancient nuclear protease2009In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 8, no 6, p. 844-851Article in journal (Refereed)
    Abstract [en]

    The nuclear proteasome activator REGgamma/PA28gamma is an ATP- and ubiquitin-independent activator of the 20S proteasome and has been proposed to degrade and thereby regulate both a key human oncogene, encoding the coactivator SRC-3/AIB1, and the cyclin-dependent kinase inhibitor p21 (Waf/Cip1). We report the identification and characterization of a PA28/REG homolog in Dictyostelium. Association of a recombinant Dictyostelium REG with the purified Dictyostelium 20S proteasome led to the preferential stimulation of the trypsin-like proteasome peptidase activity. Immunolocalization studies demonstrated that the proteasome activator is localized to the nucleus and is present in growing as well as starving Dictyostelium cells. Our results indicate that the Dictyostelium PA28/REG activator can stimulate both the trypsin-like and chymotrypsin-like activities of the 20S proteasome and supports the idea that the REGgamma-20S proteasome represents an early unique nuclear degradation pathway for eukaryotic cells.

  • 11. Popova, Blagovesta
    et al.
    Kuhlmann, Markus
    Hinas, Andrea
    Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences Box 590, S-75124 Uppsala, Sweden .
    Söderbom, Fredrik
    Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences Box 590, S-75124 Uppsala, Sweden .
    Nellen, Wolfgang
    HelF, a putative RNA helicase acts as a nuclear suppressor of RNAi but not antisense mediated gene silencing2006In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no 3, p. 773-784Article in journal (Refereed)
    Abstract [en]

    We have identified a putative RNA helicase from Dictyostelium that is closely related to drh-1, the 'dicer-related-helicase' from Caenorhabditis elegans and that also has significant similarity to proteins from vertebrates and plants. Green fluorescent protein (GFP)-tagged HelF protein was localized in speckles in the nucleus. Disruption of the helF gene resulted in a mutant morphology in late development. When transformed with RNAi constructs, HelF- cells displayed enhanced RNA interference on four tested genes. One gene that could not be knocked-down in the wild-type background was efficiently silenced in the mutant. Furthermore, the efficiency of silencing in the wild-type was dramatically improved when helF was disrupted in a secondary transformation. Silencing efficiency depended on transcription levels of hairpin RNA and the threshold was dramatically reduced in HelF- cells. However, the amount of siRNA did not depend on hairpin transcription. HelF is thus a natural nuclear suppressor of RNA interference. In contrast, no improvement of gene silencing was observed when mutant cells were challenged with corresponding antisense constructs. This indicates that RNAi and antisense have distinct requirements even though they may share parts of their pathways.

  • 12. Sandrini, Michael Paolo Bastner
    et al.
    Söderbom, Fredrik
    Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences, Box 590, SE-75124 Uppsala, Sweden.
    Mikkelsen, Nils Egil
    Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences, Box 590, SE-75124 Uppsala, Sweden.
    Piskur, Jure
    Dictyostelium discoideum salvages purine deoxyribonucleosides by highly specific bacterial-like deoxyribonucleoside kinases2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 369, no 3, p. 653-664Article in journal (Refereed)
    Abstract [en]

    The salvage of deoxyribonucleosides in the social amoeba Dictyostelium discoideum, which has an extremely A+T-rich genome, was investigated. All native deoxyribonucleosides were phosphorylated by D. discoideum cell extracts and we subcloned three deoxyribonucleoside kinase (dNK) encoding genes. D. discoideum thymidine kinase was similar to the human thymidine kinase 1 and was specific for thymidine with a K(m) of 5.1 microM. The other two cloned kinases were phylogenetically closer to bacterial deoxyribonucleoside kinases than to the eukaryotic enzymes. D. discoideum deoxyadenosine kinase (DddAK) had a K(m) for deoxyadenosine of 22.7 microM and a k(cat) of 3.7 s(-1) and could not efficiently phosphorylate any other native deoxyribonucleoside. D. discoideum deoxyguanosine kinase was also a purine-specific kinase and phosphorylated significantly only deoxyguanosine, with a K(m) of 1.4 microM and a k(cat) of 3 s(-1). The two purine-specific deoxyribonucleoside kinases could represent ancient enzymes present in the common ancestor of bacteria and eukaryotes but remaining only in a few eukaryote lineages. The narrow substrate specificity of the D. discoideum dNKs reflects the biased genome composition and we attempted to explain the strict preference of DddAK for deoxyadenosine by modeling the active center with different substrates. Apart from its native substrate, deoxyadenosine, DddAK efficiently phosphorylated fludarabine. Hence, DddAK could be used in the enzymatic production of fludarabine monophosphate, a drug used in the treatment of chronic lymphocytic leukemia.

  • 13.
    Schmith, Anika
    et al.
    Univ Jena, Inst Pharm, Dept Pharmaceut Biol, D-07743 Jena, Germany..
    Spaller, Thomas
    Univ Jena, Inst Pharm, Dept Pharmaceut Biol, D-07743 Jena, Germany..
    Gaube, Friedemann
    Univ Jena, Inst Pharm, Dept Pharmaceut Biol, D-07743 Jena, Germany..
    Fransson, Asa
    Swedish Univ Agr Sci, Biomed Ctr, Dept Mol Biol, Uppsala, Sweden..
    Boesler, Benjamin
    Univ Kassel, Inst Biol Genet, D-34125 Kassel, Germany..
    Ojha, Sandeep
    Jacobs Univ Bremen, Ribogenet Biochem Lab, Dept Life Sci & Chem, Mol Life Sci Res Ctr, D-28759 Bremen, Germany..
    Nellen, Wolfgang
    Univ Kassel, Inst Biol Genet, D-34125 Kassel, Germany..
    Hammann, Christian
    Jacobs Univ Bremen, Ribogenet Biochem Lab, Dept Life Sci & Chem, Mol Life Sci Res Ctr, D-28759 Bremen, Germany..
    Söderbom, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Winckler, Thomas
    Univ Jena, Inst Pharm, Dept Pharmaceut Biol, D-07743 Jena, Germany..
    A host factor supports retrotransposition of the TRE5-A population in Dictyostelium cells by suppressing an Argonaute protein2015In: Mobile DNA, ISSN 1759-8753, E-ISSN 1759-8753, Vol. 6, article id 14Article in journal (Refereed)
    Abstract [en]

    Background: In the compact and haploid genome of Dictyostelium discoideum control of transposon activity is of particular importance to maintain viability. The non-long terminal repeat retrotransposon TRE5-A amplifies continuously in D. discoideum cells even though it produces considerable amounts of minus-strand (antisense) RNA in the presence of an active RNA interference machinery. Removal of the host-encoded C-module-binding factor (CbfA) from D. discoideum cells resulted in a more than 90 % reduction of both plus-and minus-strand RNA of TRE5-A and a strong decrease of the retrotransposition activity of the cellular TRE5-A population. Transcriptome analysis revealed an approximately 230-fold overexpression of the gene coding for the Argonaute-like protein AgnC in a CbfA-depleted mutant. Results: The D. discoideum genome contains orthologs of RNA-dependent RNA polymerases, Dicer-like proteins, and Argonaute proteins that are supposed to represent RNA interference pathways. We analyzed available mutants in these genes for altered expression of TRE5-A. We found that the retrotransposon was overexpressed in mutants lacking the Argonaute proteins AgnC and AgnE. Because the agnC gene is barely expressed in wild-type cells, probably due to repression by CbfA, we employed a new method of promoter-swapping to overexpress agnC in a CbfA-independent manner. In these strains we established an in vivo retrotransposition assay that determines the retrotransposition frequency of the cellular TRE5-A population. We observed that both the TRE5-A steady-state RNA level and retrotransposition rate dropped to less than 10 % of wild-type in the agnC overexpressor strains. Conclusions: The data suggest that TRE5-A amplification is controlled by a distinct pathway of the Dictyostelium RNA interference machinery that does not require RNA-dependent RNA polymerases but involves AgnC. This control is at least partially overcome by the activity of CbfA, a factor derived from the retrotransposon's host. This unusual regulation of mobile element activity most likely had a profound effect on genome evolution in D. discoideum.

  • 14.
    Söderbom, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Small Nucleolar RNAs: Identification, Structure,and Function2006In: Small RNAs: Analysis and Regulatory Functions / [ed] Wolfgang Nellen and Christian Hammann, Springer, 2006, p. 31-56Chapter in book (Other academic)
    Abstract [en]

    The revelation in the last few years of a large number of new noncoding RNAs (ncRNAs) has revolutionized our view of how gene regulation works. This is to a large extent due to the recent advances in computational as well as experimental methodology, combined with an increasing number of sequenced genomes which have had an enormous impact on the quest for new small ncRNAs. These RNAs have a function per se and are not merely intermediates in the transfer of information from genes to proteins. Instead they have turned out to be involved in the regulation of many complex biological processes, including stress response, cell differentiation, and even in control of diseases. This chapter briefly describes some of the methods used to identify and isolate different classes of ncRNAs in the size range 50–500 nt. One of these, small nucleolar RNAs, will be discussed in detail.

  • 15.
    Söderbom, Fredrik
    et al.
    Center for Molecular Genetics, Department of Biology, University of California San Diego, La Jolla, CA 92093, USA.
    Anjard, C
    Iranfar, N
    Fuller, D
    Loomis, W F
    An adenylyl cyclase that functions during late development of Dictyostelium1999In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 126, no 23, p. 5463-5471Article in journal (Refereed)
    Abstract [en]

    A variety of extracellular signals lead to the accumulation of cAMP which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA(-) mutant cells can be rescued by developing them in mixtures with wild-type cells, suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI) we discovered a novel adenylyl cyclase gene, acrA, that is expressed at low levels in growing cells and at more than 25-fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA(-) mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation, plateaus during the slug stage and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA(-) cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk.

  • 16.
    Söderbom, Fredrik
    et al.
    Center for Molecular Genetics, Dept of Biology, University of California San Diego, La Jolla, CA 92093, USA.
    Loomis, W F
    Cell-cell signaling during Dictyostelium development1998In: Trends in Microbiology, ISSN 0966-842X, E-ISSN 1878-4380, Vol. 6, no 10, p. 402-406Article in journal (Refereed)
    Abstract [en]

    Specific proteins and peptides, as well as cAMP, are used as intercellular signals in Dictyostelium. Our understanding of the signal transduction pathways activated by these signals has been expanded by inclusion of newly characterized proteins. cAMP-dependent protein kinase (PKA) and its associated phosphodiesterase, RegA, play multiple roles in these pathways.

  • 17.
    Söderbom, Fredrik
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Mikrobiologi.
    Kirsebom, Leif A
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Mikrobiologi.
    RNase E cleavage in the 5' leader of a tRNA precursor.2005In: J Mol Biol, ISSN 0022-2836, Vol. 352, no 1, p. 22-7Article in journal (Other scientific)
  • 18. van Biesen, T
    et al.
    Söderbom, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, E G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Frost, L S
    Structural and functional analyses of the FinP antisense RNA regulatory system of the F conjugative plasmid1993In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 10, no 1, p. 35-43Article in journal (Refereed)
    Abstract [en]

    The efficiency of conjugation of F-like plasmids is regulated by the FinOP fertility inhibition system. The transfer (tra) operon is under the direct control of the TraJ transcriptional activator which, in turn, is negatively regulated by FinP, an antisense RNA, and FinO, a 22 kDa protein. Recently, FinO has been shown to extend the chemical stability of FinP in vivo in the absence of traJ mRNA. The in vitro secondary structures of both the FinP and TraJ RNAs were determined by the use of single- and double-strand-specific nucleases; both RNAs were found to have double stem-loop structures that are complementary to each other and, therefore, FinP RNA and TraJ RNA have the potential to form a duplex with each other. This was verified by in vitro binding experiments. The reaction was shown to be biomolecular with an apparent rate constant (kapp) of 5 x 10(5)M-1s-1, a value that is similar to those found for other natural antisense RNA systems. Preliminary evidence for the in vivo formation of the FinP-TraJ RNA duplex was obtained by primer extension of the traJ mRNA; the presence of both FinO and FinP was required to cause a dramatic reduction in the steady-state level of traJ mRNA, perhaps as a result of RNase III degradation of the resulting RNA duplex.

  • 19. Vetukuri, Ramesh R.
    et al.
    Asman, Anna K. M.
    Tellgren-Roth, Christian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Jahan, Sultana N.
    Reimegard, Johan
    Fogelqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Plant Ecology and Evolution.
    Savenkov, Eugene
    Söderbom, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Avrova, Anna O.
    Whisson, Stephen C.
    Dixelius, Christina
    Evidence for Small RNAs Homologous to Effector-Encoding Genes and Transposable Elements in the Oomycete Phytophthora infestans2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 12, p. e51399-Article in journal (Refereed)
    Abstract [en]

    Phytophthora infestans is the oomycete pathogen responsible for the devastating late blight disease on potato and tomato. There is presently an intense research focus on the role(s) of effectors in promoting late blight disease development. However, little is known about how they are regulated, or how diversity in their expression may be generated among different isolates. Here we present data from investigation of RNA silencing processes, characterized by non-coding small RNA molecules (sRNA) of 19-40 nt. From deep sequencing of sRNAs we have identified sRNAs matching numerous RxLR and Crinkler (CRN) effector protein genes in two isolates differing in pathogenicity. Effector gene-derived sRNAs were present in both isolates, but exhibited marked differences in abundance, especially for CRN effectors. Small RNAs in P. infestans grouped into three clear size classes of 21, 25/26 and 32 nt. Small RNAs from all size classes mapped to RxLR effector genes, but notably 21 nt sRNAs were the predominant size class mapping to CRN effector genes. Some effector genes, such as PiAvr3a, to which sRNAs were found, also exhibited differences in transcript accumulation between the two isolates. The P. infestans genome is rich in transposable elements, and the majority of sRNAs of all size classes mapped to these sequences, predominantly to long terminal repeat (LTR) retrotransposons. RNA silencing of Dicer and Argonaute genes provided evidence that generation of 21 nt sRNAs is Dicer-dependent, while accumulation of longer sRNAs was impacted by silencing of Argonaute genes. Additionally, we identified six microRNA (miRNA) candidates from our sequencing data, their precursor sequences from the genome sequence, and target mRNAs. These miRNA candidates have features characteristic of both plant and metazoan miRNAs. Citation: Vetukuri RR, Asman AKM, Tellgren-Roth C, Jahan SN, Reimegard J, et al. (2012) Evidence for Small RNAs Homologous to Effector-Encoding Genes and Transposable Elements in the Oomycete Phytophthora infestans.

  • 20. Wang, Nancy
    et al.
    Söderbom, Fredrik
    Center for Molecular Genetics, Department of Biology, University of California—San Diego, La Jolla, California .
    Anjard, Christophe
    Shaulsky, Gad
    Loomis, William F
    SDF-2 induction of terminal differentiation in Dictyostelium discoideum is mediated by the membrane-spanning sensor kinase DhkA.1999In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 19, no 7, p. 4750-4756Article in journal (Refereed)
    Abstract [en]

    SDF-2 is a peptide released by prestalk cells during culmination that stimulates prespore cells to encapsulate. Genetic evidence indicates that the response is dependent on the dhkA gene. This gene encodes a member of the histidine kinase family of genes that functions in two-component signal transduction pathways. The sequence of the N-terminal half of DhkA predicts two hydrophobic domains separated by a 310-amino-acid loop that could bind a ligand. By inserting MYC6 epitopes into DhkA, we were able to show that the loop is extracellular while the catalytic domain is cytoplasmic. Cells expressing the MYC epitope in the extracellular domain of DhkA were found to respond only if induced with 100-fold-higher levels of SDF-2 than required to induce dhkA+ cells; however, they could be induced to sporulate by addition of antibodies specific to the MYC epitope. To examine the enzymatic activity of DhkA, we purified the catalytic domain following expression in bacteria and observed incorporation of labelled phosphate from ATP consistent with histidine autophosphorylation. Site-directed mutagenesis of histidine1395 to glutamine in the catalytic domain blocked autophosphorylation. Furthermore, genetic analyses showed that histidine1395 and the relay aspartate2075 of DhkA are both critical to its function but that another histidine kinase, DhkB, can partially compensate for the lack of DhkA activity. Sporulation is drastically reduced in double mutants lacking both DhkA and DhkB. Suppressor studies indicate that the cyclic AMP (cAMP) phosphodiesterase RegA and the cAMP-dependent protein kinase PKA act downstream of DhkA.

  • 21. Wiegand, Stephan
    et al.
    Meier, Doreen
    Seehafer, Carsten
    Malicki, Marek
    Hofmann, Patrick
    Schmith, Anika
    Winckler, Thomas
    Foeldesi, Balint
    Boesler, Benjamin
    Nellen, Wolfgang
    Reimegard, Johan
    Kaeller, Max
    Haellman, Jimmie
    Emanuelsson, Olof
    Avesson, Lotta
    Söderbom, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hammann, Christian
    The Dictyostelium discoideum RNA-dependent RNA polymerase RrpC silences the centromeric retrotransposon DIRS-1 post-transcriptionally and is required for the spreading of RNA silencing signals2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 5, p. 3330-3345Article in journal (Refereed)
    Abstract [en]

    Dictyostelium intermediate repeat sequence 1 (DIRS-1) is the founding member of a poorly characterized class of retrotransposable elements that contain inverse long terminal repeats and tyrosine recombinase instead of DDE-type integrase enzymes. In Dictyostelium discoideum, DIRS-1 forms clusters that adopt the function of centromeres, rendering tight retrotransposition control critical to maintaining chromosome integrity. We report that in deletion strains of the RNA-dependent RNA polymerase RrpC, full-length and shorter DIRS-1 messenger RNAs are strongly enriched. Shorter versions of a hitherto unknown long non-coding RNA in DIRS-1 antisense orientation are also enriched in rrpC(-) strains. Concurrent with the accumulation of long transcripts, the vast majority of small (21 mer) DIRS-1 RNAs vanish in rrpC(-) strains. RNASeq reveals an asymmetric distribution of the DIRS-1 small RNAs, both along DIRS-1 and with respect to sense and antisense orientation. We show that RrpC is required for post-transcriptional DIRS-1 silencing and also for spreading of RNA silencing signals. Finally, DIRS-1 mis-regulation in the absence of RrpC leads to retrotransposon mobilization. In summary, our data reveal RrpC as a key player in the silencing of centromeric retrotransposon DIRS-1. RrpC acts at the post-transcriptional level and is involved in spreading of RNA silencing signals, both in the 5' and 3' directions.

1 - 21 of 21
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