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All Roads Lead to the Non-Coding RNome: Evolution of Multicellularity and Host Response to Bacterial Infection
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2020. , p. 56
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1934
Keywords [en]
ncRNA, Dictyostelia, miRNA, Class I RNA, host-pathogen interaction
National Category
Evolutionary Biology Microbiology
Research subject
Biology with specialization in Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-408825ISBN: 978-91-513-0946-0 (print)OAI: oai:DiVA.org:uu-408825DiVA, id: diva2:1423841
Public defence
2020-06-05, Room A1:111a, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2020-05-13 Created: 2020-04-15 Last updated: 2020-05-13
List of papers
1. Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum
Open this publication in new window or tab >>Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum
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2018 (English)In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 15, no 7, p. 937-954Article in journal (Refereed) Published
Abstract [en]

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

Place, publisher, year, edition, pages
UK: Taylor & Francis Group, 2018
Keywords
Dicer, microRNA, amoeba, biogenesis, evolution, transcriptional start site, intron, development, Dictyostelium discoideum
National Category
Microbiology
Research subject
Biology
Identifiers
urn:nbn:se:uu:diva-354016 (URN)10.1080/15476286.2018.1481697 (DOI)000445654400013 ()
Funder
Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, 621-2013-4665Carl Tryggers foundation , CST12:485
Available from: 2018-06-19 Created: 2018-06-19 Last updated: 2020-04-15Bibliographically approved
2. Abundantly expressed class of non-coding RNAs conserved through the multicellular evolution of dictyostelid social amoebae
Open this publication in new window or tab >>Abundantly expressed class of non-coding RNAs conserved through the multicellular evolution of dictyostelid social amoebae
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

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.

Keywords
social amoeba, Dictyostelium discoideum, non-coding RNA, evolution, Class I RNA, dictyostelids, multicellularity, Dictyostelia
National Category
Evolutionary Biology
Identifiers
urn:nbn:se:uu:diva-408822 (URN)
Funder
Carl Tryggers foundation , CST 18:381
Available from: 2020-04-15 Created: 2020-04-15 Last updated: 2020-04-15
3. Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model
Open this publication in new window or tab >>Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model
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2019 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 20, article id 961Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
BMC, 2019
Keywords
Host-pathogen, Infection, High-throughput sequencing, Mycobacteria, Legionella, Dictyostelium discoideum, Macrophage, Infection model, Pathogenic bacteria, Intracellular pathogen
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-404710 (URN)10.1186/s12864-019-6269-x (DOI)000508019700003 ()31823727 (PubMedID)
Funder
Swedish Research Council, 621-2013-4665Swedish Research Council Formas, 221-2008-580
Available from: 2020-02-26 Created: 2020-02-26 Last updated: 2020-04-15Bibliographically approved
4. Mycobacterial infection induces specific tRNA cleavage in the host cell – a response conserved from amoebae to macrophages
Open this publication in new window or tab >>Mycobacterial infection induces specific tRNA cleavage in the host cell – a response conserved from amoebae to macrophages
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

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.

Keywords
intracellular infection, Dictyostelium, microRNA, tRNA halves
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-408824 (URN)
Funder
Swedish Research Council, 2013-4665Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, 221–2008-580
Available from: 2020-04-15 Created: 2020-04-15 Last updated: 2020-04-15

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