| 1. |
- Kjellin, Jonas, et al.
(författare)
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Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model
- 2019
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 20:1
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Tidskriftsartikel (refereegranskat)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.
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| 2. |
- Liao, Zhen, 1983-
(författare)
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A small amoeba at the crossroads of the big RNAi world : MicroRNA biogenesis and Argonaute function in Dictyostelium discoideum
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
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| 3. |
- Liao, Zhen, 1983-, et al.
(författare)
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Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum
- 2018
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Ingår i: RNA Biology. - UK : Taylor & Francis Group. - 1547-6286 .- 1555-8584. ; 15:7, s. 937-954
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Tidskriftsartikel (refereegranskat)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.
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| 4. |
- Schmith, Anika, et al.
(författare)
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A host factor supports retrotransposition of the TRE5-A population in Dictyostelium cells by suppressing an Argonaute protein
- 2015
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Ingår i: Mobile DNA. - : Springer Science and Business Media LLC. - 1759-8753. ; 6
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Tidskriftsartikel (refereegranskat)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.
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| 5. |
- Sterk, Maaike M.
(författare)
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Ribosome standby sites and other structural aspects of translation initiation regions in Escherichia coli
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
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