SwePub
Sök i SwePub databas

  Extended search

Träfflista för sökning "WFRF:(Söderbom Fredrik) "

Search: WFRF:(Söderbom Fredrik)

  • Result 1-50 of 52
Sort/group result
   
EnumerationReferenceCoverFind
1.
  • Alm Rosenblad, Magnus, 1957, et al. (author)
  • The Signal Recognition Particle of the diplomonad Giardia lacks the Alu domain responsible for translational arrest
  • 2008
  • In: RNA Society Meeting 2008.
  • Conference paper (other academic/artistic)abstract
    • One of the most conserved cellular processes is the co-translational targeting of secretory and membrane proteins to the Sec translocon by the Signal Recognition Particle (SRP). This ribonucleoprotein particle consists in most eukaryotes of one RNA molecule and six proteins, and may be divided into two domains with distinct functions: the "S domain", which is most conserved, binds to the nascent peptide chain as it emerges from the exit tunnel of the ribosome, and the "Alu domain" which has a translation-regulatory function and causes an elongation arrest of the peptide chain. Of the six proteins only two is part of the Alu domain: SRP9/14.
  •  
2.
  • Anjard, Christophe, et al. (author)
  • Requirements for the adenylyl cyclases in the development of Dictyostelium
  • 2001
  • In: Development. - 0950-1991 .- 1477-9129. ; 128:18, s. 3649-3654
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
3.
  • Aspegren, Anders, et al. (author)
  • Novel non-coding RNAs in Dictyostelium discoideum and their expression during development
  • 2004
  • In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 32:15, s. 4646-4656
  • Journal article (peer-reviewed)abstract
    • The quest for non-coding RNAs (ncRNAs) in the last few years has revealed a surprisingly large number of small RNAs belonging to previously known as well as entirely novel classes. Computational and experimental approaches have uncovered new ncRNAs in all kingdoms of life. In this work, we used a shotgun cloning approach to construct full-length cDNA libraries of small RNAs from the eukaryotic model organism Dictyostelium discoideum. Interestingly, two entirely novel classes of RNAs were identified of which one is developmentally regulated. The RNAs within each class share conserved 5'- and 3'-termini that can potentially form stem structures. RNAs of both classes show predominantly cytoplasmic localization. In addition, based on conserved structure and/or sequence motifs, several of the identified ncRNAs could be divided into classes known from other organisms, e.g. 18 small nucleolar RNA candidates (17 box C/D, of which a few are developmentally regulated, and one box H/ACA). Two ncRNAs showed a high degree of similarity to the small nuclear U2 RNA and signal recognition particle RNA (SRP RNA), respectively. Furthermore, the majority of the regions upstream of the sequences encoding the isolated RNAs share conserved motifs that may constitute new promoter elements.
  •  
4.
  • Avesson, Lotta, et al. (author)
  • Abundant class of non-coding RNA regulates development in the social amoeba Dictyostelium discoideum
  • 2011
  • In: RNA Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 8:6, s. 1094-1104
  • Journal article (peer-reviewed)abstract
    • Non-coding (nc)RNAs are important players in most biological processes. Although small RNAs such as microRNAs and small interfering RNAs have emerged as exceptionally important regulators of gene expression, great numbers of larger ncRNAs have also been identified. Many of these are abundant and differentially expressed but their functions have in most cases not been elucidated. The social amoeba Dictyostelium discoideum contain the ncRNAs commonly found in eukaryotes. In addition, we previously reported the identification of two novel classes of 42-65 nt long stem-loop forming RNAs, Class I and Class II RNAs, with unknown function. In this study we have further characterized these abundant ncRNAs, which are down regulated during development. We have confirmed expression of 29 Class I RNAs and experimentally verified the formation of the computationally predicted short conserved stem structure. Furthermore, we have for the first time created knockout strains for several small ncRNA genes in D. discoideum and found that deletion of one of the Class I RNAs, DdR-21, results in aberrant development. In addition we have shown that this Class I RNA forms a complex with one or several proteins but do not appear to be associated with ribosomes or polysomes. In a pull down assay, several proteins interacting with DdR-21 were identified, one of these has two RNA recognition motifs (RRMs). The purified RRM containing protein was demonstrated to bind directly and specifically to DdR-21.
  •  
5.
  • Avesson, Lotta, et al. (author)
  • MicroRNAs in Amoebozoa : Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs
  • 2012
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 18:10, s. 1771-1782
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
6.
  • B. Moreno, Anaísa (author)
  • Evolution and host-specific adaptations of Legionella pneumophila
  • 2022
  • Doctoral thesis (other academic/artistic)abstract
    • How bacteria evolve pathogenic traits is shaped by their communities and environments. Legionella pneumophila is ubiquitous in aquatic habitats, where it persists by replicating within a broad range of protozoan hosts. Using the same mechanisms, L. pneumophila may also accidentally infect humans, causing a severe pneumonia known as Legionnaires’ disease. As hosts, humans are evolutionary dead-ends, resulting in the loss of human-specific adaptations after infection. This thesis aims to identify and characterise these host adaptations.In Paper I, we study the in-patient evolution of L. pneumophila. We collected a large set of strains from sporadic infections and outbreaks, pairing clinical isolates with their respective environmental sources. Using comparative genomic analyses, we identified two genes individually mutated in three independent infections. One gene encoded an outer membrane protein, a homolog from the OmpP1/FadL family, and the other an EAL domain-containing protein. These results suggest that convergent evolution may be at play and that these mutations are potential candidates for human-specific host adaptations.In Paper II, we investigate host adaptation and the selective pressures that drive it using a long-term experimental evolution approach. We passaged L. pneumophila in Acanthamoeba castellanii and U937 macrophages, separately and in alternation, for over 800 generations. We found 49 fixed mutations across the 18 evolved populations: two distinct mutations in RpsL, which confers streptomycin resistance, as well as two additional mutations, each consistently associated with one of the former, in the chaperonin GroES or in RpsD, a known compensatory mutation. Mutations in the lipopolysaccharide synthesis operon were observed only in lineages passaged in A. castellanii, whilst mutations in LerC were fixed in six lineages passaged in U937, making these candidate mutations for host-specific adaptations.In Paper III, we shift focus to A. castellanii, a natural host of L. pneumophila. We describe a novel method for high-efficiency transfection of this amoeba with a cationic polymer. Using a systematic approach to test different parameters, we found that widely available and inexpensive polyethylenimines can be used to transfect A. castellanii at a much greater efficiency than the currently used reagents.In conclusion, these studies suggest that although L. pneumophila can infect humans, it is sub-optimally adapted for it, and offer potential determinants of host-specificity in L. pneumophila.
  •  
7.
  • Berggren, Sofia, et al. (author)
  • ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis
  • 2024
  • In: mSphere. - : American Society for Microbiology. - 2379-5042. ; 9:3
  • Journal article (peer-reviewed)abstract
    • Gastrointestinal disease caused by Salmonella enterica is associated with the pathogen's ability to replicate within epithelial cells and macrophages. Upon host cell entry, the bacteria express a type-three secretion system encoded within Salmonella pathogenicity island 2, through which host-manipulating effector proteins are secreted to establish a stable intracellular niche. Transcription of this intracellular virulence program is activated by the PhoPQ two-component system that senses the low pH and the reduced magnesium concentration of host cell vacuoles. In addition to transcriptional control, Salmonella commonly employ RNA-binding proteins (RBPs) and small regulatory RNAs (sRNAs) to regulate gene expression at the post-transcriptional level. ProQ is a globally acting RBP in Salmonella that promotes expression of the intracellular virulence program, but its RNA repertoire has previously been characterized only under standard laboratory growth conditions. Here, we provide a high-resolution ProQ interactome during conditions mimicking the environment of the Salmonella-containing vacuole (SCV), revealing hundreds of previously unknown ProQ binding sites in sRNAs and mRNA 3 ' UTRs. ProQ positively affected both the levels and the stability of many sRNA ligands, some of which were previously shown to associate with the well-studied and infection-relevant RBP Hfq. We further show that ProQ activates the expression of PhoP at the post-transcriptional level, which, in turn, leads to upregulation of the intracellular virulence program.
  •  
8.
  • Boesler, Carsten, et al. (author)
  • Sequence and generation of mature ribosomal RNA transcripts in Dictyostelium discoideum
  • 2011
  • In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 286:20, s. 17693-17703
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
9.
  • Crona, Mikael, et al. (author)
  • A Rare Combination of Ribonucleotide Reductases in the Social Amoeba Dictyostelium discoideum
  • 2013
  • In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:12, s. 8198-8208
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
10.
  •  
11.
  • Diesend, Jan, et al. (author)
  • Fractional 2 '-O-methylation in the ribosomal RNA of Dictyostelium discoideum supports ribosome heterogeneity in Amoebozoa
  • 2022
  • In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 12:1
  • Journal article (peer-reviewed)abstract
    • A hallmark of ribosomal RNA (rRNA) are 2 '-O-methyl groups that are introduced sequence specifically by box C/D small nucleolar RNAs (snoRNAs) in ribonucleoprotein particles. Most data on this chemical modification and its impact on RNA folding and stability are derived from organisms of the Opisthokonta supergroup. Using bioinformatics and RNA-seq data, we identify 30 novel box C/D snoRNAs in Dictyostelium discoideum, many of which are differentially expressed during the multicellular development of the amoeba. By applying RiboMeth-seq, we find 49 positions in the 17S and 26S rRNA 2 '-O-methylated. Several of these nucleotides are substoichiometrically modified, with one displaying dynamic modification levels during development. Using homology-based models for the D. discoideum rRNA secondary structures, we localize many modified nucleotides in the vicinity of the ribosomal A, P and E sites. For most modified positions, a guiding box C/D snoRNA could be identified, allowing to determine idiosyncratic features of the snoRNA/rRNA interactions in the amoeba. Our data from D. discoideum represents the first evidence for ribosome heterogeneity in the Amoebozoa supergroup, allowing to suggest that it is a common feature of all eukaryotes.
  •  
12.
  •  
13.
  • Edelbroek, Bart, et al. (author)
  • Evolution of microRNAs in Amoebozoa and implications for the origin of multicellularity
  • 2024
  • In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 52:6, s. 3121-3136
  • Journal article (peer-reviewed)abstract
    • MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression in both plants and animals. They are thought to have evolved convergently in these lineages and hypothesized to have played a role in the evolution of multicellularity. In line with this hypothesis, miRNAs have so far only been described in few unicellular eukaryotes. Here, we investigate the presence and evolution of miRNAs in Amoebozoa, focusing on species belonging to Acanthamoeba, Physarum and dictyostelid taxonomic groups, representing a range of unicellular and multicellular lifestyles. miRNAs that adhere to both the stringent plant and animal miRNA criteria were identified in all examined amoebae, expanding the total number of protists harbouring miRNAs from 7 to 15. We found conserved miRNAs between closely related species, but the majority of species feature only unique miRNAs. This shows rapid gain and/or loss of miRNAs in Amoebozoa, further illustrated by a detailed comparison between two evolutionary closely related dictyostelids. Additionally, loss of miRNAs in the Dictyostelium discoideum drnB mutant did not seem to affect multicellular development and, hence, demonstrates that the presence of miRNAs does not appear to be a strict requirement for the transition from uni- to multicellular life.
  •  
14.
  • Edelbroek, Bart, et al. (author)
  • Evolution of microRNAs in Amoebozoa and implications for the origin of multicellularity
  • 2024
  • In: Nucleic Acids Research. - 0305-1048 .- 1362-4962.
  • Journal article (peer-reviewed)abstract
    • MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression in both plants and animals. They are thought to have evolved convergently in these lineages and hypothesized to have played a role in the evolution of multicellularity. In line with this hypothesis, miRNAs have so far only been described in few unicellular eukaryotes. Here, we investigate the presence and evolution of miRNAs in Amoebozoa, focusing on species belonging to Acanthamoeba, Physarum and dictyostelid taxonomic groups, representing a range of unicellular and multicellular lifestyles. miRNAs that adhere to both the stringent plant and animal miRNA criteria were identified in all examined amoebae, expanding the total number of protists harbouring miRNAs from 7 to 15. We found conserved miRNAs between closely related species, but the majority of species feature only unique miRNAs. This shows rapid gain and/or loss of miRNAs in Amoebozoa, further illustrated by a detailed comparison between two evolutionary closely related dictyostelids. Additionally, loss of miRNAs in the Dictyostelium discoideum drnB mutant did not seem to affect multicellular development and, hence, demonstrates that the presence of miRNAs does not appear to be a strict requirement for the transition from uni- to multicellular life.
  •  
15.
  • Edelbroek, Bart (author)
  • Function and Evolution of Small Regulatory RNAs and their Associated Proteins : A Journey from Genome to Proteome
  • 2024
  • Doctoral thesis (other academic/artistic)abstract
    • Organisms throughout the tree of life have evolved distinct ways to regulate gene expression. Some of these processes involve non-coding RNAs (ncRNAs), which are not translated but functional nonetheless. These ncRNAs are of utmost importance, with dysregulation of some causing severe developmental effects or even being lethal.In order to get a better fundamental understanding of gene regulation, and the ncRNAs that evolved to regulate gene expression, we study this in Amoebozoa. Members of this taxon vary greatly in lifestyle and organismal complexity. Some are strictly unicellular, free-living, whereas others, such as the social amoeba Dictyostelium discoideum can transition between unicellular and multicellular lifestyles. D. discoideum features a variety of small ncRNAs. Among these are the microRNAs. microRNAs have mostly been studied in plants and animals, where they are believed to have evolved convergently, and hypothesized to have played a role when these taxa evolved multicellular lifestyles. At what point the D. discoideum microRNAs evolved, how they function, and if they are involved in its multicellular lifestyle are fundamental questions addressed in this thesis. Here, we studied the evolution and function of microRNAs in a broad set of species belonging to Amoebozoa. We could identify microRNAs in all studied amoebae, and concluded that they are probably not involved in the evolution of multicellularity. To in detail investigate the evolution of microRNAs, we performed comparative genomics using D. discoideum and the close relative Dictyostelium firmibasis. For this, we sequenced, assembled and annotated the genome of the latter. At this point, our findings suggest that the microRNAs evolved several times in Amoebozoa, although we cannot rule out if they have a deep evolutionary history.The Class I RNAs are another type of ncRNAs. These, on the other hand, are only present in the social amoebae. They are hypothesized to regulate the transition from unicellular to multicellular in these species, potentially in a post-transcriptional manner. In order to investigate this, it is essential to understand to what extent the proteome and transcriptome correlate. Hence, we performed paired transcriptomics and proteomics in a time-series during multicellular development. By including a strain in which a specific Class I RNA is knocked out, we have initiated studies of its role during the transition to multicellularity.In conclusion, we were able to answer broad evolutionary and functional questions about gene regulation and ncRNAs by studying Amoebozoa from genome to proteome. 
  •  
16.
  •  
17.
  • He, Lin, et al. (author)
  • PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of ColE1-related plasmids
  • 1993
  • In: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 9:6, s. 1131-1142
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
18.
  • Hinas, Andrea, et al. (author)
  • Identification of the Major Spliceosomal RNAs in Dictyostelium discoideum Reveals Developmentally Regulated U2 Variants and Polyadenylated snRNAs
  • 2006
  • In: Eukaryotic Cell. - 1535-9778 .- 1535-9786. ; 5:6, s. 924-934
  • Journal article (peer-reviewed)abstract
    • Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5' cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5' end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm.
  •  
19.
  • Hinas, Andrea, et al. (author)
  • The small RNA repertoire of Dictyostelium discoideum and its regulation by components of the RNAi pathway
  • 2007
  • In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 35:20, s. 6714-6726
  • Journal article (peer-reviewed)abstract
    • Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 1826 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase.
  •  
20.
  • Hinas, Andrea, et al. (author)
  • Treasure hunt in an amoeba : non-coding RNAs in Dictyostelium discoideum.
  • 2007
  • In: Current Genetics. - : Springer Science and Business Media LLC. - 0172-8083 .- 1432-0983. ; 51:3, s. 141-159
  • Journal article (peer-reviewed)abstract
    • The traditional view of RNA being merely an intermediate in the transfer of genetic information, as mRNA, spliceosomal RNA, tRNA, and rRNA, has become outdated. The recent discovery of numerous regulatory RNAs with a plethora of functions in biological processes has truly revolutionized our understanding of gene regulation. Tiny RNAs such as microRNAs and small interfering RNAs play vital roles at different levels of gene control. Small nucleolar RNAs are much more abundant than previously recognized, and new functions beyond processing and modification of rRNA have recently emerged. Longer non-coding RNAs (ncRNAs) can also have important regulatory roles in the cell, e.g., antisense RNAs that control their target mRNAs. The majority of these important findings arose from analyses in various model organisms. In this review, we focus on ncRNAs in the social amoeba Dictyostelium discoideum. This important genetically tractable model organism has recently received renewed attention in terms of discovery, regulation and functional studies of ncRNAs. Old and recent findings are discussed and put in context of what we today know about ncRNAs in other organisms.
  •  
21.
  • Hällman, Jimmie, et al. (author)
  • Identification and verification of microRNAs by high-throughput sequencing
  • 2013. - 2
  • In: Dictyostelium discoideum Protocols. - New York : Humana Press. - 9781627033015 ; , s. 125-138
  • Book chapter (peer-reviewed)abstract
    • 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.
  •  
22.
  • Kjellin, Jonas, et al. (author)
  • Abundantly expressed class of non-coding RNAs conserved through the multicellular evolution of dictyostelid social amoebae
  • Other publication (other academic/artistic)abstract
    • Background: Aggregative multicellularity has evolved multiple times in diverse groups of eukaryotes. One of the most well-studied examples is the development of dictyostelid social amoebae, e.g. Dictyostelium discoideum. However, it is still poorly understood why multicellularity emerged in these amoebae while the great majority of other members of Amoebozoa are unicellular. Previously a novel type of non-coding RNA, Class I RNAs, was identified in D. discoideum and demonstrated to be important for normal multicellular development. In this study we investigated Class I RNA evolution and its connection to multicellular development.Results: New Class I RNA genes were identified by constructing a co-variance model combined with a scoring system based on conserved up-stream sequences. Multiple genes were predicted in representatives of each major group of Dictyostelia and expression analysis validated that our search approach can identify expressed Class I RNA genes with high accuracy and sensitivity. Further studies showed that Class I RNAs are ubiquitous in Dictyostelia and share several highly conserved structure and sequence motifs. Class I RNA genes appear to be unique to dictyostelid social amoebae since they could not be identified in searches in outgroup genomes, including the closest known relatives to Dictyostelia.Conclusion: Our results show that Class I RNA is an ancient abundant class of ncRNAs, likely to have been present in the last common ancestor of Dictyostelia dating back at least 600 million years. Taken together, our current knowledge of Class I RNAs suggests that they may have been involved in evolution of multicellularity in Dictyostelia.
  •  
23.
  • Kjellin, Jonas, et al. (author)
  • Abundantly expressed class of noncoding RNAs conserved through the multicellular evolution of dictyostelid social amoebas
  • 2021
  • In: Genome Research. - : Cold Spring Harbor Laboratory Press (CSHL). - 1088-9051 .- 1549-5469. ; 31:3, s. 436-447
  • Journal article (peer-reviewed)abstract
    • Aggregative multicellularity has evolved multiple times in diverse groups of eukaryotes, exemplified by the well-studied development of dictyostelid social amoebas, for example, Dictyostelium discoideum. However, it is still poorly understood why multicellularity emerged in these amoebas while the majority of other members of Amoebozoa are unicellular. Previously, a novel type of noncoding RNA, Class I RNAs, was identified in D. discoideum and shown to be important for normal multicellular development. Here, we investigated Class I RNA evolution and its connection to multicellular development. We identified a large number of new Class I RNA genes by constructing a covariance model combined with a scoring system based on conserved upstream sequences. Multiple genes were predicted in representatives of each major group of Dictyostelia and expression analysis confirmed that our search approach identifies expressed Class I RNA genes with high accuracy and sensitivity and that the RNAs are developmentally regulated. Further studies showed that Class I RNAs are ubiquitous in Dictyostelia and share highly conserved structure and sequence motifs. In addition, Class I RNA genes appear to be unique to dictyostelid social amoebas because they could not be identified in outgroup genomes, including their closest known relatives. Our results show that Class I RNA is an ancient class of ncRNAs, likely to have been present in the last common ancestor of Dictyostelia dating back at least 600 million years. Based on previous functional analyses and the presented evolutionary investigation, we hypothesize that Class I RNAs were involved in evolution of multicellularity in Dictyostelia.
  •  
24.
  • Kjellin, Jonas, 1986- (author)
  • All Roads Lead to the Non-Coding RNome : Evolution of Multicellularity and Host Response to Bacterial Infection
  • 2020
  • Doctoral thesis (other academic/artistic)abstract
    • The ability to control gene expression is fundamental for all living organisms. Therefore, a large variety of regulatory mechanisms exist in each cell which are essential for e.g. developmental processes and to quickly adapt to different cellular stresses such as infection. Today we know that much of this regulation depends on non-coding (nc)RNAs. However, the function and evolutionary origin of many ncRNAs remains to be understood.The work presented in this thesis revolves around the evolutionary group of Dictyostelia. These social amoebae grow as single cells but initiate a multicellular development program when food runs low. The evolutionary position of Dictyostelia within Amoebozoa together with their multicellular development make these organisms relevant for investigating the evolution of ncRNAs and their association with multicellularity. Furthermore, the dictyostelid Dictyostelium discoideum is one of few organisms besides plants and animals were miRNAs have been identified. It is also an established model organism, well-adapted for laboratory growth and detailed molecular work.In this thesis, we investigate the biogenesis of miRNAs in D. discoideum and show that the Dicer-like protein DrnB is essential for global miRNA maturation. Next, we study the evolution of another ncRNA, Class I RNAs, and show that these are conserved in all dictyostelids and likely emerged in their last common ancestor. Lastly, we utilize the D. discoideum infection model to study the regulation of messenger RNAs and ncRNAs upon infection by Mycobacterium marinum and Legionella pneumophila to improve our understanding of the complex interactions between host and pathogen. We show that the two bacteria induce distinct mRNA regulation in D. discoideum. In addition, we detected high levels of specific tRNA halves generated in the host in response to M. marinum but not L. pneumophila or bacteria utilized as food. Despite the large evolutionary distances, the regulation of both mRNAs and ncRNAs in D. discoideum was, in many aspects, representative for the regulation in macrophages after infection.In conclusion, by using a seemingly simple group of organisms, social amoebae, this thesis work addresses major questions such as the role of ncRNA in multicellular evolution and the intricate host-pathogen interplay during bacterial infection.
  •  
25.
  • Kjellin, Jonas, et al. (author)
  • Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model
  • 2019
  • In: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 20:1
  • Journal article (peer-reviewed)abstract
    • Background: During infection by intracellular pathogens, a highly complex interplay occurs between the infected cell trying to degrade the invader and the pathogen which actively manipulates the host cell to enable survival and proliferation. Many intracellular pathogens pose important threats to human health and major efforts have been undertaken to better understand the host-pathogen interactions that eventually determine the outcome of the infection. Over the last decades, the unicellular eukaryote Dictyostelium discoideum has become an established infection model, serving as a surrogate macrophage that can be infected with a wide range of intracellular pathogens. In this study, we use high-throughput RNA-sequencing to analyze the transcriptional response of D. discoideum when infected with Mycobacterium marinum and Legionella pneumophila. The results were compared to available data from human macrophages.Results: The majority of the transcriptional regulation triggered by the two pathogens was found to be unique for each bacterial challenge. Hallmark transcriptional signatures were identified for each infection, e.g. induction of endosomal sorting complexes required for transport (ESCRT) and autophagy genes in response to M. marinum and inhibition of genes associated with the translation machinery and energy metabolism in response to L. pneumophila. However, a common response to the pathogenic bacteria was also identified, which was not induced by non-pathogenic food bacteria. Finally, comparison with available data sets of regulation in human monocyte derived macrophages shows that the elicited response in D. discoideum is in many aspects similar to what has been observed in human immune cells in response to Mycobacterium tuberculosis and L. pneumophila.Conclusions: Our study presents high-throughput characterization of D. discoideum transcriptional response to intracellular pathogens using RNA-seq. We demonstrate that the transcriptional response is in essence distinct to each pathogen and that in many cases, the corresponding regulation is recapitulated in human macrophages after infection by mycobacteria and L. pneumophila. This indicates that host-pathogen interactions are evolutionary conserved, derived from the early interactions between free-living phagocytic cells and bacteria. Taken together, our results strengthen the use of D. discoideum as a general infection model.
  •  
26.
  • Kjellin, Jonas, et al. (author)
  • Mycobacterial infection induces specific tRNA cleavage in the host cell – a response conserved from amoebae to macrophages
  • Other publication (other academic/artistic)abstract
    • Intracellular bacterial pathogens have to avoid the defenses of the host cell and create an environment in which they can replicate. This causes complex host-pathogen interactions, which are not fully understood. We have previously shown that infection by Mycobacterium marinum and Legionella pneumophila, respectively, induce large transcriptional rewiring in the model amoeba Dictyostelium discoideum. Although the major part of the responses was unique to each pathogen, both infections caused an up-regulation of RNA interference (RNAi) associated genes. Comparison to regulation in human macrophages after infection with Mycobacterium tuberculosis and L. pneumophila indicated that parts of the host-pathogen interaction, including the regulation of RNAi associated genes, are conserved.In this study, we investigate the effect on the small RNA population in D. discoideum when the amoeba was infected with M. marinum and L. pneumophila, respectively. Similar to the regulation of protein coding genes, we show that the two pathogens cause very different small RNA responses. M. marinum infection causes a dramatic up-regulation of specific tRNA halves, which was not observed in response to L. pneumophila or Klebsiella pneumonia. Furthermore, we show that this response is conserved in mammalian cells after infection by mycobacteria while L. pneumophila infection, as in D. discoideum, does not cause an increase in tRNA-halves.In summary, we demonstrate that infection by M. marinum induces major changes in the small RNA population of D. discoideum. This response is characterized by cleavage of specific host tRNAs, generating high levels of tRNA-halves, and is conserved in macrophages infected by M. tuberculosis.
  •  
27.
  • Kuhlmann, Markus, et al. (author)
  • Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi
  • 2005
  • In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 33:19, s. 6405-6417
  • Journal article (peer-reviewed)abstract
    • We have identified a DNA methyltransferase of the Dnmt2 family in Dictyostelium that was denominated DnmA. Expression of the dnmA gene is downregulated during the developmental cycle. Overall DNA methylation in Dictyostelium is approximately 0.2% of the cytosine residues, which indicates its restriction to a limited set of genomic loci. Bisulfite sequencing of specific sites revealed that DnmA is responsible for methylation of mostly asymmetric C-residues in the retrotransposons DIRS-1 and Skipper. Disruption of the gene resulted in a loss of methylation and in increased transcription and mobilization of Skipper. Skipper transcription was also upregulated in strains that had genes encoding components of the RNA interference pathway disrupted. In contrast, DIRS-1 expression was not affected by a loss of DnmA but was strongly increased in strains that had the RNA-directed RNA polymerase gene rrpC disrupted. A large number of siRNAs were found that corresponded to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA modification. No siRNAs corresponding to the standard Skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene silencing in Dictyostelium but that different, partially overlapping mechanisms control transposon silencing.
  •  
28.
  • Kyriakopoulou, Christina, et al. (author)
  • U1-like snRNAs lacking complementarity to canonical 5' splice sites
  • 2006
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 12:9, s. 1603-1611
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
29.
  • Larsson, Pontus, et al. (author)
  • De novo search for non-coding RNA genes in the AT-rich genome of Dictyostelium discoideum : Performance of Markov-dependent genome feature scoring
  • 2008
  • In: Genome Research. - : Cold Spring Harbor Laboratory. - 1088-9051 .- 1549-5469. ; 18:6, s. 888-899
  • Journal article (peer-reviewed)abstract
    • Genome data are increasingly important in the computational identification of novel regulatory non-coding RNAs (ncRNAs). However, most ncRNA gene-finders are either specialized to well-characterized ncRNA gene families or require comparisons of closely related genomes. We developed a method for de novo screening for ncRNA genes with a nucleotide composition that stands out against the background genome based on a partial sum process. We compared the performance when assuming independent and first-order Markov-dependent nucleotides, respectively, and used Karlin-Altschul and Karlin-Dembo statistics to evaluate the significance of hits. We hypothesized that a first-order Markov-dependent process might have better power to detect ncRNA genes since nearest-neighbor models have been shown to be successful in predicting RNA structures. A model based on a first-order partial sum process (analyzing overlapping dinucleotides) had better sensitivity and specificity than a zeroth-order model when applied to the AT-rich genome of the amoeba Dictyostelium discoideum. In this genome, we detected 94% of previously known ncRNA genes (at this sensitivity, the false positive rate was estimated to be 25% in a simulated background). The predictions were further refined by clustering candidate genes according to sequence similarity and/or searching for an ncRNA-associated upstream element. We experimentally verified six out of 10 tested ncRNA gene predictions. We conclude that higher-order models, in combination with other information, are useful for identification of novel ncRNA gene families in single-genome analysis of D. discoideum. Our generalizable approach extends the range of genomic data that can be searched for novel ncRNA genes using well-grounded statistical methods.
  •  
30.
  • Liao, Zhen, 1983- (author)
  • A small amoeba at the crossroads of the big RNAi world : MicroRNA biogenesis and Argonaute function in Dictyostelium discoideum
  • 2018
  • Doctoral thesis (other academic/artistic)abstract
    • Small non-coding RNA (ncRNA) mediated gene silencing, known as RNAi, is a key regulatory mechanism of gene expression in eukaryotes. MicroRNAs (miRNA), one major type of small ncRNAs, are about 21nt long and bound by Argonaute proteins. This RNA-protein complex, called RISC, silences post-transcriptionally target mRNAs containing partial or full complementary sequence to the miRNA.  MiRNAs are generated from step-wise endonucleolytic cleavages of long primary transcripts (pri-miRNAs) by RNase III nucleases. Biogenesis of miRNAs differs between uni- and multicellular eukaryotes, and also between plants and animals. In this thesis, I aimed to understand miRNA maturation in the social amoeba Dictyostelium discoideum, which stands at the crossroads between these phylogenetically distant groups. We showed that Dicer-like protein DrnB is essential for global maturation of D. discoideum miRNAs. The study of two pri-miRNAs revealed the conserved 5’ m7G-cap structures, but different 3’end formation from each other, and also from canonical miRNAs in plants and animals. In agreement with its evolutionary position, D. discoideum miRNA biogenesis showed unique and also shared features with both life groups.D. discoideum grows as a unicellular organism, but can switch to a multicellular development upon starvation. Most miRNAs, many other small ncRNAs, and Argonaute proteins, the core effectors of the RISC, are differentially expressed during development, indicative of a crucial role of RNAi mediated regulation throughout D. discoideum life cycle. Among the five Argonaute homologs in D. discoideum, I investigated the functions of three members, e.g. AgnB, C and E. Judging from their subcellular localization, the phenotypic consequences and transcriptional alteration resulting from single Argonaute gene deletion, our results suggested different roles of AgnB, C and E. Possibly AgnB associates with miRNAs and regulates gene expression post-transcriptionally; while AgnC seems to be involved in nuclear RNAi. Finally, the cytoplasmic AgnE inhibits D. discoideum cell growth and regulates developmental timing via an unknown mechanism.My thesis work expands our knowledge on D. discoideum RNAi with focuses on miRNA biogenesis and potential function of Argonaute proteins and, all together, sheds lights on the evolution of miRNA and RNAi.  
  •  
31.
  • Liao, Zhen, 1983-, et al. (author)
  • Functional analyses of RNA interference effectors in Dictyostelium discoideum during growth and development
  • Other publication (other academic/artistic)abstract
    • 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.
  •  
32.
  • Liao, Zhen, 1983-, et al. (author)
  • Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum
  • 2018
  • In: RNA Biology. - UK : Taylor & Francis Group. - 1547-6286 .- 1555-8584. ; 15:7, s. 937-954
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
33.
  • Liao, Zhen, 1983-, et al. (author)
  • The Dictyostelium discoideum Argonaute protein AgnE regulates cell growth
  • Other publication (other academic/artistic)abstract
    • 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.
  •  
34.
  • Masson, Patrick, et al. (author)
  • Characterization of a REG/PA28 Proteasome Activator Homolog in Dictyostelium discoideum Indicates that the Ubiquitin- and ATP-Independent REGγ Proteasome Is an Ancient Nuclear Protease
  • 2009
  • In: Eukaryotic Cell. - 1535-9778 .- 1535-9786. ; 8:6, s. 844-851
  • Journal article (peer-reviewed)abstract
    • The nuclear proteasome activator REGγ/PA28γ 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 REGγ-20S proteasome represents an early unique nuclear degradation pathway for eukaryotic cells.
  •  
35.
  •  
36.
  • Popova, Blagovesta, et al. (author)
  • HelF, a putative RNA helicase acts as a nuclear suppressor of RNAi but not antisense mediated gene silencing
  • 2006
  • In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 34:3, s. 773-784
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
37.
  • Ramesh, Vetukuri, et al. (author)
  • Evidence for involvement of Dicer-like, Argonaute and histone deacetylase proteins in gene silencing in Phytophthora infestans
  • 2011
  • In: Molecular Plant Pathology. - 1464-6722 .- 1364-3703. ; 12, s. 772-785
  • Journal article (peer-reviewed)abstract
    • Gene silencing may have a direct or indirect impact on many biological processes in eukaryotic cells, and is a useful tool for the determination of the roles of specific genes. In this article, we report silencing in Phytophthora infestans, an oomycete pathogen of potato and tomato. Gene silencing is known to occur in P. infestans, but its genetic basis has yet to be determined. Genes encoding the major components of the RNA interference (RNAi) pathway, Dicer-like (Pidcl1), Argonaute (Piago1-5) and RNA-directed RNA polymerase (Pirdr1), were identified in the P. infestans genome by comparative genomics, together with families of other genes potentially involved in gene silencing, such as histone deacetylases, histone methyltransferases, DEAD heli-cases, chromodomain proteins and a class 1 RNaseIII. Real-time reverse transcription-polymerase chain reaction demonstrated transcript accumulation for all candidate genes throughout the asexual lifecycle and plant infection, but at different levels of mRNA abundance. A functional assay was developed in which silencing of the sporulation-associated Picdc14 gene was released by the treatment of protoplasts with in vitro-synthesized double-stranded RNAs homologous to Pidcl1, Piago1/2 and histone deacetylase Pihda1. These results suggest that the components of gene silencing, namely Dicer-like, Argonaute and histone deacetylase, are functional in P. infestans. Our data demonstrate that this oomycete possesses canonical gene silencing pathways similar to those of other eukaryotes.
  •  
38.
  • Ramesh, Vetukuri, et al. (author)
  • Evidence for Small RNAs Homologous to Effector-Encoding Genes and Transposable Elements in the Oomycete Phytophthora infestans
  • 2012
  • In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:12, s. e51399-
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
39.
  • Ringqvist, Emma, 1979-, et al. (author)
  • Transcriptional changes in Giardia during host-parasite interactions
  • 2011
  • In: International Journal for Parasitology. - : Elsevier BV. - 0020-7519 .- 1879-0135. ; 41:3-4, s. 277-285
  • Journal article (peer-reviewed)abstract
    • Giardia intestinalis is one of the major causes of parasite-induced diarrhea. The disease, giardiasis, is caused by trophozoites attaching to the intestinal epithelium, resulting in apoptosis of intestinal epithelial cells, disrupted epithelial barrier function and malabsorption. Microarray studies have detected gene expression changes in intestinal epithelial cells (IECs), during interaction with Giardia trophozoites in vitro. In the present study we examined this host-parasite interaction further by transcriptional profiling of interacting trophozoites using Giardia oligo microarrays. A total of 325 Giardia transcripts were significantly changed during the interaction, lasting 1.5 to 18 hrs in complete DMEM medium. Quantitative real-time reverse transcriptase PCR confirmed these changes in all 12 genes tested using mRNA isolated in a separate experiment. Genes involved in cell division and attachment were down-regulated in the late time-points of interaction. Proteins earlier suggested to be important during host parasite interactions like arginine deiminase and cysteine proteinases changed their expression. Oxygen defense proteins and several members of the high cysteine-rich membrane protein (HCMp) family were up-regulated during the interaction with IECs. Thus, there are extensive gene-expression changes in Giardia trophozoites and IECs during host-parasite interactions and this can be important for establishment of infection and in the induction of giardiasis.
  •  
40.
  • Rizvanovic, Alisa, et al. (author)
  • Saturation mutagenesis charts the functional landscape of Salmonella ProQ and reveals a gene regulatory function of its C-terminal domain
  • 2021
  • In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 49:17, s. 9992-10006
  • Journal article (peer-reviewed)abstract
    • The global RNA-binding protein ProQ has emerged as a central player in post-transcriptional regulatory networks in bacteria. While the N-terminal domain (NTD) of ProQ harbors the major RNA-binding activity, the role of the ProQ C-terminal domain (CTD) has remained unclear. Here, we have applied saturation mutagenesis coupled to phenotypic sorting and long-read sequencing to chart the regulatory capacity of Salmonella ProQ. Parallel monitoring of thousands of ProQ mutants allowed mapping of critical residues in both the NTD and the CTD, while the linker separating these domains was tolerant to mutations. Single amino acid substitutions in the NTD associated with abolished regulatory capacity strongly align with RNA-binding deficiency. An observed cellular instability of ProQ associated with mutations in the NTD suggests that interaction with RNA protects ProQ from degradation. Mutation of conserved CTD residues led to overstabilization of RNA targets and rendered ProQ inert in regulation, without affecting protein stability in vivo. Furthermore, ProQ lacking the CTD, although binding competent, failed to protect an mRNA target from degradation. Together, our data provide a comprehensive overview of residues important for ProQ-dependent regulation and reveal an essential role for the enigmatic ProQ CTD in gene regulation.
  •  
41.
  • Sandrini, Michael, et al. (author)
  • Dictyostelium discoideum salvages purine deoxyribonucleosides by highly specific bacterial-like deoxyribonucleoside kinases
  • 2007
  • In: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 369:3, s. 653-664
  • Journal article (peer-reviewed)abstract
    • 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 mu M. 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 mu M 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 mu M 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.
  •  
42.
  • Schmith, Anika, et al. (author)
  • A host factor supports retrotransposition of the TRE5-A population in Dictyostelium cells by suppressing an Argonaute protein
  • 2015
  • In: Mobile DNA. - : Springer Science and Business Media LLC. - 1759-8753. ; 6
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
43.
  • Sterk, Maaike M. (author)
  • Ribosome standby sites and other structural aspects of translation initiation regions in Escherichia coli
  • 2018
  • Doctoral thesis (other academic/artistic)abstract
    • Translation initiation, which is rate-limiting in protein synthesis, is often the step at which regulation occurs. Here, we investigated several mechanisms of translation initiation in Escherichia coli, including their control. First, we showed that translation of the transcriptional regulator CsgD is inhibited by two sRNAs through a direct antisense mechanism.In some bacterial mRNAs, the ribosome binding site (RBS) is sequestered in a stable structure, which generally generates very low protein output. Yet, these mRNAs are often efficiently translated, which suggested the requirement for “ribosome standby sites”. Here, we investigated the structure and sequence features of an effective standby site using plasmid-borne GFP reporter constructs, and showed that relatively short, single-stranded regions near a structurally sequestered RBS can profoundly increase translation rates. Both the length and the sequence of these single-stranded regions are important for standby site efficiency, and the standby site needs to be single-stranded. This work serves as a proof-of-principle study of the ribosome standby model.To investigate the sequence-dependency of standby sites further, we used an unbiased approach, creating plasmid libraries containing millions of different standby sites in the same reporter plasmid as before. Cells were sorted by fluorescence according to translational levels, and standby sites analyzed by deep sequencing. This analysis showed that efficient standby sites have a low GC-content and rarely contain Shine-Dalgarno sequences. Additionally, nucleotides near the 3’-border of the standby region affect translation efficiency more than those closer to the 5’-end. Mutational and structure-probing experiments are planned to verify these findings.
  •  
44.
  • Söderbom, Fredrik, et al. (author)
  • An adenylyl cyclase that functions during late development of Dictyostelium
  • 1999
  • In: Development. - 0950-1991 .- 1477-9129. ; 126:23, s. 5463-5471
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
45.
  • Söderbom, Fredrik, et al. (author)
  • Cell-cell signaling during Dictyostelium development
  • 1998
  • In: Trends in Microbiology. - 0966-842X .- 1878-4380. ; 6:10, s. 402-406
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
46.
  •  
47.
  • Söderbom, Fredrik (author)
  • Small Nucleolar RNAs : Identification, Structure,and Function
  • 2006
  • In: Small RNAs. - Berlin, Heidelberg : Springer. - 9783540281306 ; , s. 31-56
  • Book chapter (other academic/artistic)abstract
    • 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.
  •  
48.
  • Söderbom, Fredrik (author)
  • Structure, function and metabolic stability of antisense RNAs
  • 1997
  • Doctoral thesis (other academic/artistic)abstract
    • Antisense RNAs usually are small, highly structured, unstable RNAs that control a varietyof different biological processes. They exert their effect by binding to complementarytarget RNAs and thereby inhibit their functions.This thesis is concerned with the main features that determine the efficiency of antisense RNAs: their metabolic stability, the binding rate between antisense and target RNA, and the structure of the antisense RNA itself. Three different plasmid-encoded antisense RNAs were studied.RNA decay in bacteria is complex: a large number of proteins are involved in processing and degradation. My results show that mutations in an Escherichia coli gene, pcnB, encoding a poly(A)polymerase, exert an effect on the replication of antisense RNA-regulated plasmids. PcnB aids in the degradation of the inhibitors of replication, the antisense RNAs. This enzyme can polyadenylate both CopA and RNAI, the antisense RNAs controlling the replication of plasmids R1 and ColE1, respectively. Accelerated pcnB dependent decay of CopA requires processing by an endoribonuclease, RNase E. This cleavage step appears to initiate decay of both CopA and RNA I. In addition, the 3'-5' exoribonucleases PNPase and RNase II can independently degrade RNase E-cleaved CopA.Other ribonucleases are involved in the pcnB dependent degradation since CopA decay is shown (although at lower rates) in the absence of these enzymes.Analysis of FinP, an antisense RNA controlling plasmid conjugation, indicates astructure consisting of two stem-loops. Stem-loops are preferred motifs in most antisenseRNAs. The binding rate between FinP and its target RNA is in the same range as in otherwell-characterized systems. Upon binding, the target RNA (traJ mRNA) is renderedunstable, probably due to RNase III-dependent cleavages. This destabilization requiresthe presence of the helper protein FinO.
  •  
49.
  • van Biesen, T, et al. (author)
  • Structural and functional analyses of the FinP antisense RNA regulatory system of the F conjugative plasmid
  • 1993
  • In: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 10:1, s. 35-43
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
50.
  • Wang, Nancy, et al. (author)
  • SDF-2 induction of terminal differentiation in Dictyostelium discoideum is mediated by the membrane-spanning sensor kinase DhkA.
  • 1999
  • In: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 19:7, s. 4750-4756
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
Skapa referenser, mejla, bekava och länka
  • Result 1-50 of 52
Type of publication
journal article (34)
other publication (8)
doctoral thesis (7)
book chapter (2)
conference paper (1)
Type of content
peer-reviewed (34)
other academic/artistic (18)
Author/Editor
Söderbom, Fredrik (48)
Avesson, Lotta (10)
Hinas, Andrea (8)
Reimegård, Johan (7)
Larsson, Pontus (5)
Kirsebom, Leif A. (4)
show more...
Ramesh, Vetukuri (2)
Lundin, Daniel, 1965 ... (2)
Grabherr, Manfred (2)
Svärd, Staffan (2)
Dixelius, Christina (2)
Sahlin, Margareta (2)
Sjöberg, Britt-Marie (2)
Holmqvist, Erik, 197 ... (2)
Anjard, Christophe (2)
Loomis, William F (2)
Biryukova, Inna (2)
Friedländer, Marc R. (2)
Käller, Max (2)
Fransson, Åsa (2)
Larsson, Anders (1)
Liu, Lei (1)
Bergquist, Jonas (1)
Hellman, Ulf (1)
Alm Rosenblad, Magnu ... (1)
Andersson, Dan I. (1)
Fuller, D (1)
Piskur, Jure (1)
Emanuelsson, Olof (1)
Nielsen, Henrik (1)
Ardell, David H. (1)
Wagner, E.G.H. (1)
Tellgren-Roth, Chris ... (1)
Koskiniemi, Sanna (1)
Wagner, Gerhart E. H ... (1)
Sandrini, Michael (1)
Jerlström-Hultqvist, ... (1)
Svärd, Staffan G. (1)
Fogelqvist, Johan (1)
Andresen, Liis (1)
Rizvanovic, Alisa (1)
Aspegren, Anders (1)
Isberg, Ralph R. (1)
Schumacher, Heiko T (1)
Fechter, Pierre (1)
Romby, Pascale (1)
B. Moreno, Anaísa (1)
Guy, Lionel, PhD, Do ... (1)
Ensminger, Alexander ... (1)
Akusjärvi, Göran, Pr ... (1)
show less...
University
Uppsala University (47)
Swedish University of Agricultural Sciences (10)
Stockholm University (7)
Royal Institute of Technology (4)
Linnaeus University (2)
University of Gothenburg (1)
show more...
Lund University (1)
show less...
Language
English (51)
Undefined language (1)
Research subject (UKÄ/SCB)
Natural sciences (49)
Medical and Health Sciences (7)
Agricultural Sciences (2)

Year

Kungliga biblioteket hanterar dina personuppgifter i enlighet med EU:s dataskyddsförordning (2018), GDPR. Läs mer om hur det funkar här.
Så här hanterar KB dina uppgifter vid användning av denna tjänst.

 
pil uppåt Close

Copy and save the link in order to return to this view