ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2020

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1934

All Roads Lead to the Non-CodingRNome

Evolution of Multicellularity and Host Response toBacterial Infection

JONAS KJELLIN

ISSN 1651-6214ISBN 978-91-513-0946-0urn:nbn:se:uu:diva-408825

Dissertation presented at Uppsala University to be publicly examined in Room A1:111a,Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, Friday, 5 June 2020 at 13:00 forthe degree of Doctor of Philosophy. The examination will be conducted in English. Facultyexaminer: Professor Jonathan Chubb (Medical Research Council Laboratory for MolecularCell Biology, University College London).

AbstractKjellin, J. 2020. All Roads Lead to the Non-Coding RNome. Evolution of Multicellularityand Host Response to Bacterial Infection. Digital Comprehensive Summaries of UppsalaDissertations from the Faculty of Science and Technology 1934. 56 pp. Uppsala: ActaUniversitatis Upsaliensis. ISBN 978-91-513-0946-0.

The ability to control gene expression is fundamental for all living organisms. Therefore, a largevariety of regulatory mechanisms exist in each cell which are essential for e.g. developmentalprocesses and to quickly adapt to different cellular stresses such as infection. Today we knowthat much of this regulation depends on non-coding (nc)RNAs. However, the function andevolutionary 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 programwhen food runs low. The evolutionary position of Dictyostelia within Amoebozoa together withtheir multicellular development make these organisms relevant for investigating the evolution ofncRNAs and their association with multicellularity. Furthermore, the dictyostelid Dictyosteliumdiscoideum is one of few organisms besides plants and animals were miRNAs have beenidentified. It is also an established model organism, well-adapted for laboratory growth anddetailed molecular work.

In this thesis, we investigate the biogenesis of miRNAs in D. discoideum and show that theDicer-like protein DrnB is essential for global miRNA maturation. Next, we study the evolutionof another ncRNA, Class I RNAs, and show that these are conserved in all dictyostelidsand likely emerged in their last common ancestor. Lastly, we utilize the D. discoideuminfection model to study the regulation of messenger RNAs and ncRNAs upon infection byMycobacterium marinum and Legionella pneumophila to improve our understanding of thecomplex interactions between host and pathogen. We show that the two bacteria induce distinctmRNA regulation in D. discoideum. In addition, we detected high levels of specific tRNA halvesgenerated in the host in response to M. marinum but not L. pneumophila or bacteria utilizedas food. Despite the large evolutionary distances, the regulation of both mRNAs and ncRNAsin D. discoideum was, in many aspects, representative for the regulation in macrophages afterinfection.

In conclusion, by using a seemingly simple group of organisms, social amoebae, this thesiswork addresses major questions such as the role of ncRNA in multicellular evolution and theintricate host-pathogen interplay during bacterial infection.

Keywords: ncRNA, Dictyostelia, miRNA, Class I RNA, host-pathogen interaction

Jonas Kjellin, Department of Cell and Molecular Biology, Microbiology, Box 596, UppsalaUniversity, SE-75124 Uppsala, Sweden.

© Jonas Kjellin 2020

ISSN 1651-6214ISBN 978-91-513-0946-0urn:nbn:se:uu:diva-408825 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-408825)

Till Johanna och Fred

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Liao, Z., Kjellin, J., Hoeppner, M.P., Grabherr, M. & Söderbom,

F. (2018). Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum. RNA Biology 15(7):937–54.

II Kjellin, J., Avesson, L., Reimegård, J., Liao, Z., Eichinger, L., Noegel, A., Glöckner, G., Schaap, P. & Söderbom F. Abundantly expressed class of non-coding RNAs conserved through the mul-ticellular development of dictyostelid social amoebae. Manu-script

III Kjellin, J., Pränting, M., Bach, F., Vaid, R., Edelbroek, B., Li, Z., Hoeppner, M.P., Grabherr, M., Isberg, R.R., Hagedorn, M. & Söderbom, F. (2019) Investigation of the host transcriptional re-sponse to intracellular bacterial infection using Dictyostelium discoideum as a host model. BMC genomics, 20, 961

IV Kjellin, J., Pränting, M., Edelbroek, B., Langseth, C.M. & Söder-bom, F. Mycobacterial infection induces specific tRNA cleavage in the host cell– a response conserved from amoebae to macro-phages. Manuscript

Articles are published under the Creative commons v4 license (https://creativecommons.org/licenses/by/4.0/).

Contents

Introduction ...............................................................................................11The eukaryotic non-coding RNome .......................................................11Dictyostelia ...........................................................................................12

Dictyostelid multicellular development .............................................13Multicellular development of D. discoideum .....................................14The D. discoideum model system ......................................................16

ncRNAs and multicellularity .................................................................17miRNA regulation in plants and animals ...............................................18

siRNA biogenesis .............................................................................18miRNA biogenesis and targeting in plants and animal .......................19miRNA regulation in D. discoideum .................................................21Origin of miRNA regulation .............................................................22

Class I RNAs and multicellularity .........................................................23Host-pathogen interactions and gene regulation .....................................24Gene regulation during infection in D. discoideum ................................25Tuberculosis .........................................................................................25

M. marinum as a model for M. tuberculosis.......................................26M. marinum infection in D. discoideum ............................................26

Legionnaires disease .............................................................................27Infectious route of L. pneumophila ....................................................27

Current investigations ................................................................................29Aim ......................................................................................................29Biogenesis of miRNAs in D. discoideum (Paper I) ................................29

DrnB is essential for biogenesis of all miRNAs .................................29Precursor transcript of miRNA are stabilized in DrnB depleted cells .30Biogenesis of miRNA-1176 and miRNA-1177..................................30

Evolution of Class I RNAs and multicellular development (Paper II) .....31Prediction of Class I RNAs in evolutionary distant organisms ...........31Construction of a Class I RNA classifier ...........................................32Class I RNAs are expressed in all groups of Dictyostelia ...................32Key features of D. discoideum Class I RNAs are highly conserved throughout Dictyostelia .....................................................................33Class I RNAs are developmentally regulated and ubiquitous in Dictyostelia ......................................................................................33

The emergence of Class I RNAs correspond to the evolution of multicellular development in Dictyostelia .........................................34Conservation of interacting proteins ..................................................34

Host mRNA response to infection by intracellular bacteria (Paper III) ...35Both M. marinum and L. pneumophila infection have a strong impact on host mRNA levels shortly after uptake ..............................35Distinct host responses detected after M. marinum and L. pneumophila infection ......................................................................35Comparison of host responses reveals potential common defense genes ................................................................................................36Similar regulation identified in infected macrophages .......................37

Infection by mycobacteria trigger cleavage of specific host tRNAs (Paper IV) .............................................................................................37

M. marinum causes specific tRNA cleavage in the host .....................37The tRNA-response to M. marinum is not restricted to the 5’ halves of tRNA-Asp ....................................................................................38Host tRNA cleavage is a conserved response to mycobacterial infection ...........................................................................................39

Concluding remarks and future perspectives ..............................................40Evolution of miRNAs and their connection to multicellularity ..........40Class I RNA function ........................................................................41Host-pathogen interaction and the non-coding RNome ......................41

Svensk sammanfattning .............................................................................43

Acknowledgements ...................................................................................46

References .................................................................................................47

Abbreviations

cAMP cyclic adenosine monophosphate CMF conditioned media factor DIF-1 differentiation inducing factor 1 dsRNA double stranded RNA DUSE Dictyostelium upstream element hpi hours post infection k.o. knock out LCA last common ancestor LCV legionella containing vacuole lncRNA long non-coding RNA MCV mycobacteria containing vacuole mRNA messenger RNA miRNA micro RNA ncRNA non-coding RNA PSF prestarvation factor RISC RNA induced silencing complex RNA-seq RNA sequencing RNAi RNA interference siRNA short interfering RNA sRNA small RNA tRNA transfer RNA WT wild type

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Introduction

Living organisms exhibit an astonishing variety in morphological phenotypes and life styles. Despite this, most eukaryotes are relatively similar on the cel-lular level. So how is this phenotypic variation possible and how can multi-cellular organisms become as complex as a human? A major contributing fac-tor is the ability to control gene expression. To be able to control which genes are expressed and at what level is fundamental for achieving cell type special-ization in multicellular organisms. Also, the ability to quickly reshape gene expression is required for organisms to adapt to changing conditions such as starvation or infection. As a consequence, all cells exhibit a wide array of gene regulatory mechanisms which can occur both on the transcriptional and post-transcriptional level as well as during and after translation. Today we know that large part of this regulation involves different types of non-coding RNAs.

The eukaryotic non-coding RNome All eukaryotic cells transcribe a wide variety of non-coding (nc)RNAs, and the full repertoire of ncRNAs in a cell constitute its ncRNome. These RNAs are extremely heterogenous in basically all aspects such as size, function and biogenesis. The only unifying characteristic is that they are not translated into proteins but carry out their functions as RNA molecules.

For a long time, the role of ncRNAs was greatly underestimated. It was believed that the primary role of RNA was to act as an intermediate carrier of information between DNA and protein. Although some classes of ncRNA were identified as early as the 1950’s and 1960’s, they were primarily believed to function in the basic processing of messenger (m)RNAs and their transla-tion into protein (Morris and Mattick, 2014). These classes included ribosomal (r)RNAs and small nucleolar (sno)RNAs involved in ribosome function and biogenesis, transfer (t)RNAs involved in translation and small nuclear (sn)RNAs involved in splicing.

Our understanding of the regulatory potential of ncRNAs have greatly in-creased over the past decades. Today we know that the function of the classical ncRNA classes extends beyond their original description. For example, much thanks to the advances of sequencing technologies, our understanding of the function of tRNAs and fragments thereof have greatly expanded during recent years (Keam and Hutvagner, 2015). Improved sequencing technologies have

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also led to the identification of a wealth of new classes of ncRNAs where the discovery of micro (mi)RNAs in diverse organisms have had the greatest im-pact on our view about the role of regulatory ncRNAs in eukaryotes (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001).

In the work presented in this thesis, we aim to improve our understanding of the evolution and function of two types of ncRNAs, i.e. miRNAs and Class I RNAs, by studying them in the evolutionary group of Dictyostelia. All mem-bers of Dictyostelia have the ability to transition between uni-and multicellu-larity which also allows us to investigate the connection between these ncRNAs and multicellularity. In addition, we use the dictyostelid Dictyoste-lium discoideum, which is an established model organism, to performed more detailed studies into miRNA biogenesis as well as the role of mRNA regula-tion and ncRNAs during intracellular bacterial infection.

Dictyostelia The dictyostelid social amoebae (Dictyostelia) form a monophyletic group within the Amoebozoa supergroup (Schilde et al., 2019) and their last com-mon ancestor (LCA) is estimated to date back at least 600 million years (Heidel et al., 2011). These social amoebae are unicellular as long as they have enough bacteria or other microorganisms to prey on for nutrients. When food runs low, all members of Dictyostelia are able to initiate a multicellular devel-opment where cells aggregate and eventually form multicellular fruiting bod-ies. These fruiting bodies contain spores which can be dispersed by the wind to new hunting grounds where the amoebae can reinitiate unicellular growth (Kawabe et al., 2019).

Dictyostelia has traditionally been divided into three taxa based on the mor-phology of their fruiting bodies, i.e. Dictyostelium, Polysphondylium and Acy-tostelium. With the advent of molecular phylogenetics, the phylogeny of Dic-tyostelia was revised into four major groups mainly based on small subunit ribosomal DNA (SSU rDNA) (Romeralo et al., 2011; Schaap et al., 2006).

Since then, at least one alternative phylogeny of Dictyostelia have been proposed (Sheikh et al., 2018). However, based on the latest and most well-supported phylogenetic analysis, Dictyostelia diverged early into two branches of which both are further divided into four major groups (Group 1-4) (Fig. 1) (Schilde et al., 2019). Several species have also been identified which cannot be placed in any of the major groups e.g. Polysphondylium vio-laceum which is intermediate to Group 3 and 4 (Schilde et al., 2019).

Several studies have provided large amounts of valuable data for evolution-ary studies in Dictyostelia. Full well-annotated genome sequences are availa-ble for representatives of each major group (Fig. 1) (Eichinger et al., 2005; Glöckner et al., 2016; Heidel et al., 2011; Sucgang et al., 2011; Urushihara et al., 2015). These are complemented by at least 10 publicly available draft

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genome sequences which represent both additional major group species as well as inter-group dictyostelids. The genomes of the closest known relatives to dictyostelids have also been sequenced, i.e. Physarum polycephalum and Protostelia spp. (Clarke et al., 2013; Hillmann et al., 2018; Loftus et al., 2005; Schaap et al., 2015) which are highly valuable when trying to resolve evolu-tionary origins of e.g. ncRNAs in Dictyostelia. In addition, transcriptomic time course data during multicellular development is available for representa-tive species of all major groups (Glöckner et al., 2016; Parikh et al., 2010; Rosengarten et al., 2015; Schilde et al., 2016).

Figure 1. Schematic phylogeny of Dictyostelia with representatives for each major group, as well as the two subgroups of Group 2 (Schilde et al., 2019). The placement of Amoebozoa in the eukaryotic tree of life is based on (Burki et al., 2020). Sche-matic drawings to the right denote developmental phenotype of the different organ-isms. The structures are not in scale between organisms.

Dictyostelid multicellular development In all members of Dictyostelia, the multicellular development is initiated upon starvation which induce aggregation of nearby amoebae by chemotaxis. D. discoideum and other Group 4 species, use cyclic adenosine monophosphate (cAMP) as chemoattractant (Romeralo et al., 2013). This is in contrast to most species belonging to Group 1-3, where aggregation seems to depend on a di-peptide called glorin (Romeralo et al., 2013). However, there are exceptions as neopterin and folate have been identified as the chemoattractants in the two Group 3 species Dictyostelium lacteum and Dictyostelium minutum, respec-tively (de Wit and Konijn, 1983; Van Haastert et al., 1982).

The Group 4 dictyostelids also distinguish themselves from the others in their mode of aggregation. While the members of Group 4 aggregate in streams of tightly associated cells, it is common that the Group 1-3 species migrate individually and form their first cell-to-cell contact in the resulting aggregate (Romeralo et al., 2013). Once the aggregates are formed, the major-ity of the dictyostelids go through the rest of the multicellular development

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program at the same location but some species, mainly in Group 4, have the ability to form a migrating slug. This slug moves as one unit towards light via phototaxis (Romeralo et al., 2013). In nature, this is utilized to achieve fruiting body formation on the soil surface which enables efficient spore dispersal (Bonner and Lamont, 2005; Romeralo et al., 2013).

The most distinct differences during the multicellular development of dif-ferent dictyostelids are the size and morphology of the fruiting bodies. Most Group 4 species form single fruiting bodies with an unbranched stalk while several fruiting bodies with lateral branches derived from the same aggregate is common in Group 1-3 (Fig. 1) (Romeralo et al., 2013). In the majority of the species, the fruiting bodies consist of one or multiple balls of spores which are supported by a stalk of vacuolized dead cells. This relies on the speciali-zation into at least two cell types during development, i.e. stalk cells and spore cells (Schilde et al., 2014). Interestingly, in Group 2A species, e.g. Acytoste-lium subglobosum, all cells in the fruiting body becomes spores supported by an acellular cellulose stalk (Romeralo et al., 2013). This difference is also re-flected in the transcriptome of A. subglobosum, as the regulation of gene ex-pression during development is vastly different from the one in D. discoideum (Urushihara et al., 2015).

The Group 4 species are considered to exhibit the most complex multicel-lularity of the dictyostelids. Partly because of the differences discussed above, but also because the division of labour between cells during multicellularity differs compared to other dictyostelids. Multicellular structures of D. dis-coideum consist of at least four specialized cell types which are decided rela-tively early on in development. In contrast, other dictyostelids have in general only two different cell types which differentiate based on their location in the aggregate (Schilde et al., 2014). In A. subglobosum, which creates fruiting bodies with acellular stalks, the ability to undergo cell differentiation has been lost (Mohri et al., 2013).

Multicellular development of D. discoideum The vegetative growth and multicellular development of D. discoideum was described already in 1935 when Kenneth B. Raper isolated the amoeba from decaying forest leaves (Raper, 1935). Since then the development have been extensively studied and today we know more about the aggregative multicel-lularity in D. discoideum than in any other organism.

The transition from uni-to multicellularity is associated with major rewir-ing of gene expression (Rosengarten et al., 2015) and different cell types can be distinguished based on their transcriptional signature (Antolović et al., 2019). Interestingly, both RNA machinery components as well as miRNAs are developmentally regulated in D. discoideum (Avesson et al., 2011; Hinas et al., 2007; Paper I). This suggests that miRNAs are involved in reshaping the transcriptome during development.

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The development of D. discoideum can loosely be divided into three main stages which take approximately 24 hours to complete: 1) Early development, when the cells start to aggregate, 2) Mid development, when the amoebae get organized in multicellular structures and 3) Late development, when the amoebae undergo the final differentiation and form the fruiting body (Fig. 2)

Figure 2. The multicellular development of D. discoideum. During unicellular growth, the amoebae are microscopic, ~10-20 µm in diameter, while the multicellu-lar structures are visible to the naked eye. Once the fruiting body is complete it is in general ~2 mm tall.

Early development Development is initiated by starvation but also require a high enough cell den-sity in order to occur. During vegetative growth, D. discoideum constitutively secretes a glycoprotein called PSF (prestarvation factor) (Clarke et al., 1987). A high concentration of PSF in combination with starvation results in the ex-pression of the protein kinase YakA and subsequent activation of aggregation genes (Souza et al., 1998). These aggregation genes promote the production of cAMP (Schaap, 2016). Cell density is also sensed by the conditioned media factor (CMF) which is secreted at the onset of development. If a high enough concentration of CMF is detected, the G protein-coupled receptor, CarA, will promote cAMP production via activation of adenylyl cyclase ACA (Loomis, 2014). Together, this starvation induced regulation leads to secreted pulses of cAMP which causes amoebae to stream together into a cell aggregate. The

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aggregate subsequently develops into the first multicellular structure of the development, the mound, which contains up to 100 000 cells (Schaap, 2016).

Mid development The cells that eventually will form the stalk congregate at the tip of the mound and continue to express cAMP (Dormann and Weijer, 2001). The continuous cAMP secretion will attract the cells from underneath causing the mound to grow upwards to form the finger structure which has the ability to migrate as a slug if it falls over. The posterior cells of the slug secrete cAMP which in-duce differentiation of prespore cells (Alvarez-Curto et al., 2007). The pre-spore cells then start to synthesize and secrete the differentiation inducing fac-tor 1 (DIF-1) (Neumann et al., 2010; Saito et al., 2008; Thompson and Kay, 2000). This induce the differentiation of amoebae further back in the slug into two additional cell types which later will form the basal disc and the upper and lower cup of the fruiting body (Williams, 2006).

Late development In the final stage of the development, which is also known as culmination stage, stalk cell differentiation is triggered by the expression of diguanylate cyclase A (Chen and Schaap, 2012). This eventually leads to increased intra-cellular cAMP levels and the activation of the protein kinase A (PKA) which in turn induce stalk gene expression (Chen et al., 2017). The formation of the stalk begins with the creation of a stalk tube and the downward migration of prestalk cells (Jermyn and Williams, 1991). During this process the prestalk cells begin their final differentiation into stalk cells, which includes enlarge-ment via vacuolization. Simultaneously, spore maturation is induced by simi-lar pathways, i.e. increased intracellular cAMP and PKA activation, which leads to their migration upwards along the stalk as it is being formed (Schaap, 2016). In the end, the fruiting body, characterized by a ball of spores supported by thin stalk, is complete.

The D. discoideum model system Wild isolates of D. discoideum rely on phagocytosis of bacteria and other mi-croorganisms for nutrients. However, laboratory strains capable of axenic growth via macropinocytosis have been developed (Loomis, 1971; Sussman and Sussman, 1967). These axenic strains are named AX2-4 of which mainly AX2 have been used in the studies presented in this thesis. The easy growth of the amoeba in the laboratory in combination with the early development of many tools for genetic manipulations e.g. gene-knockout and targeted muta-genesis, have made D. discoideum a popular model organism for a wide vari-ety of biological questions (Müller-Taubenberger et al., 2013).

More recently, D. discoideum have emerged as an infection model. Phago-cytic cells, e.g. macrophages, are an important part of the human innate

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immune response to bacterial infection (Medzhitov, 2007) and large parts of the cellular machinery used by these cells to ingest and degrade bacteria is well-conserved also in amoebae (Dunn et al., 2018). In addition, interaction between amoebae and bacteria in nature have likely been involved in shaping pathogens intracellular survival strategies (Molmeret et al., 2005). D. dis-coideum can be infected with several different pathogens important for human health, of which the two most studied are Mycobacterium marinum (Cardenal-Muñoz et al., 2017b) and Legionella pneumophila (Swart et al., 2018).

ncRNAs and multicellularity Today there is convincing evidence that regulatory ncRNAs have been essen-tial for the evolution of animal multicellularity. For example, Piwi-interacting RNAs, which are required for mammalian germline development, appears to have evolved in Metazoa (Gaiti et al., 2017). Also, a large number of different long non-coding (lnc)RNAs have been shown to be involved in cell differen-tiation processes (Morris and Mattick, 2014). However, the connection be-tween ncRNAs and organismal complexity have largely been attributed to miRNAs. These regulatory RNAs were discovered already in 1993, when the miRNA lin-4 was shown to regulate the levels of lin-14 mRNA in the flat worm Caenorhabditis elegans (Lee et al., 1993; Wightman et al., 1993). The real breakthrough came in the early 2000’s, when the second miRNA let-7 was identified and shown to be widely conserved among animals, including humans (Pasquinelli et al., 2000; Reinhart et al., 2000). Soon afterwards, hun-dreds of miRNAs were identified in both plants and animals (Houbaviy et al., 2003; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001; Lim et al., 2003; Mourelatos et al., 2002; Reinhart et al., 2002).

Despite their small size, ~21-22 nt, miRNAs have a major impact on the transcriptome. It has been estimated that 60 % of the human genes are under miRNA control of which many are directly involved in developmental pro-cesses (Friedman et al., 2009). Furthermore, increased organismal complexity is in general positively correlated with the size of the miRNA repertoire. Taken together, this has led to the notion that miRNAs are essential to animal multicellularity (Gaiti et al., 2017). However, miRNAs are not a requirement for multicellularity per se, since many multicellular organisms, e.g. most mul-ticellular fungi, lack miRNAs (Billmyre et al., 2013). Also, the identification of miRNAs in strictly unicellular organisms, such as Chlamydomonas rein-hardtii, proves that they are not firmly associated with multicellularity (Mol-nár et al., 2007; Zhao et al., 2007). Hence, the notion that miRNAs are strictly correlated with multicellularity has been challenged. Not only because miR-NAs have been found in unicellular organisms, but also because there are in-herent problems in measuring the complexity of an organism. In addition, with the increase in identified miRNAs in various organisms, the correlation

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between number of miRNAs and organismal complexity has become more vague (Moran et al., 2017).

In contrast to the well-documented involvement of regulatory ncRNAs in animal multicellularity, not much is known about their role in organisms with aggregative multicellularity. Interestingly, D. discoideum is one of only a handful of organisms belonging to other eukaryotic lineages than plants and animals where miRNAs have been identified (Avesson et al., 2011; Hinas et al., 2007; Paper I; Moran et al., 2017). Furthermore, a novel class of ncRNAs have been identified in D. discoideum called Class I RNAs. No function has yet been determined for these RNAs, but they are developmentally regulated and the absence of one of the Class I RNAs causes aberrant multicellular de-velopment (Aspegren et al., 2004; Avesson et al., 2011).

miRNA regulation in plants and animals The two main classes of small regulatory RNAs are miRNAs and short inter-fering (si)RNA. Both classes utilize the RNAi machinery for biogenesis and function, where the key players are the RNase III enzyme Dicer and the Ar-gonaute proteins (Cerutti and Casas-Mollano, 2006). miRNAs and siRNAs both regulate gene expression by guiding the RNA induced silencing complex (RISC) via sequence complementarity between the target and the small RNA. The main components of the RISC are the Argonaute proteins, which are con-served in all domains of life (Swarts et al., 2014).

While siRNA mediated silencing is widespread among eukaryotes, miR-NAs have mainly been identified in animal and plants (Moran et al., 2017). Taken together, this has led to the notion that the RNAi machinery and siRNA silencing was present in the last eukaryotic common ancestor, where it was used in the defense against viruses and transposable elements (Obbard et al., 2009). The origin of miRNA regulation is less clear but has been suggested to have evolved from the ancient RNAi machinery (further discussed below).

siRNA biogenesis Biogenesis of siRNAs are triggered by the presence of double stranded (ds)RNA. These dsRNA can be of many different origins, e.g. viruses and transposable elements, and are processed by Dicer into several ~21 nt long siRNA duplexes. One strand of the siRNA duplex associates with an Argo-naute protein, which is guided to the target RNA by the siRNA, leading to binding between the two RNAs via perfect complementarity (Fig. 3a) (Carthew and Sontheimer, 2009). This siRNA induced regulation is referred to as RNAi and can occur both in the cytosol and the nucleus. Nuclear RNAi often silence expression by heterochromatin formation either via DNA or

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histone methylation while RNAi in the cytosol mainly induce degradation of the target RNA by cleavage (Martienssen and Moazed, 2015).

miRNA biogenesis and targeting in plants and animal In both plants and animals, miRNAs are processed from longer RNA poly-merase II transcripts harboring a stem-loop structure (pri-miRNA). These are in turn processed to form shorter stem-loop structures (pre-miRNAs). The pri-miRNAs are commonly transcribed as independent genes (Carthew and Sontheimer, 2009). However, among animals, many of the miRNAs are pro-duced from introns of protein coding genes (Rodriguez et al., 2004)

In animals, the processing from pri- to pre-miRNA occurs in the nucleus and is performed by the microprocessor complex comprised of the RNase III enzyme Drosha and the RNA-binding protein PASHA/DGCR8 (Fig. 3b). The pre-miRNA is subsequently exported to the cytoplasm by Exportin 5, where Dicer cleaves the stem-loop precursor to ~21 nt dsRNAs (Fig. 3b) (Moran et al., 2017). One strand of the duplex is then selectively loaded into the Argo-naute protein of the RISC, which also includes GW182 family proteins. In most cases, the target is then identified by partial complementarity to the miRNA, often in the 3’ untranslated regions of the mRNA (Fig. 3b) (Bartel, 2009). This partial complementarity mainly occurs between the target and the miRNA at the nucleotide position two to eight in the 5’ end of the mature miRNA, also known as the seed region (Brennecke et al., 2005; Lewis et al., 2003).

Plants do not have Drosha, instead the initial processing of the pri-miRNA in the nucleus is performed by the Dicer-like protein 1 (DCL1) in association with the RNA binding proteins SERRATE (SE) and HYPONASTIC LEAVES1 (HYL1) (Moran et al., 2017). In contrast to animals, the resulting pre-miRNA is not transported to the cytoplasm. Instead, it is further processed by DCL1 in the nucleus to generate the miRNA duplex (Fig. 3c). Both strands of the duplex are in turn methylated at the 3’ end by HUA ENHANCER1 (HEN1) and exported to the cytoplasm by HASTY, an Exportin 5 homolog (Moran et al., 2017). One strand of the duplex is then selectively loaded into the Argonaute and guide the RISC to its target mRNA often by near complete complementarity (Fig. 3c). In contrast to animal RISC, the plant counterpart does not include GW182 (Carthew and Sontheimer, 2009).

The differences in target identification in plants and animals also leads to different outcomes. In general, perfect to near-perfect complementarity be-tween the miRNA and its target lead to target degradation by Argonaute cleav-age, which have large effects on the levels of the target mRNA (Ameres and Zamore, 2013). In contrast, miRNA targeting by partial complementarity in-duce translational inhibition and/or deadenylation of the target mRNA. This also leads to reduced transcripts levels, but to a more modest degree compared to degradation by cleavage (Baek et al., 2008; Selbach et al., 2008).

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Figure 3. Biogenesis and main mode of targeting of siRNAs and miRNAs in ani-mals and plants. a) siRNA - double stranded (ds)RNA is cleaved by Dicer generat-ing many siRNA duplexes. Single stranded siRNA guide RISC to its target by per-fect complementarity. b) Animal miRNA – miRNA precursors are transcribed by RNA Pol II and processed by the microprocessor in the nucleus. The remaining stem-loop structure is transported to the cytosol by Exportin 5 where it is further processed by Dicer to the miRNA duplex. One of the strands subsequently associate with an Argonaut protein and guide the RISC to its target by partial complementa-rity. c) Plant miRNA - pri-miRNAs are transcribed by RNA Pol II and processed to the miRNA duplex by DCL-1 and associated proteins in the nucleus. The miRNAs are methylated in the 3’ end before the duplex is transported to the cytosol by HASTY. One of the strands in the duplex associate with an Argonaute protein and guide the RISC to its target by perfect or near-perfect complementarity.

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miRNA regulation in D. discoideum The sequencing of the D. discoideum genome revealed a RNAi machinery consisting of five Argonaute proteins (AgnA-E) and two Dicer-like proteins (DrnA-B) while no Drosha homologue was identified (Eichinger et al., 2005). Not long after, the first two miRNAs were discovered in D. discoideum (Hinas et al., 2007) and with the development of improved sequencing technologies, the miRNA repertoire have been expanded to approximately 30 miRNAs (Avesson et al., 2012; Paper I; Meier et al., 2016). These miRNAs only make up a minor fraction of the small RNAs in D. discoideum. Instead, the majority are 21 nt long siRNAs derived from the retro-transposable element DIRS-1 (Avesson et al., 2012; Hinas et al., 2007; Paper I).

Similar to the processing in plants, miRNA maturation in D. discoideum occurs in the nucleus and rely on the complex of DrnB and the RNA-binding protein RbdB (Paper I; Meier et al., 2016). The DrnB-RbdB complex also af-fects longer precursor transcripts of miRNAs suggesting that it is involved in both the processing of the pri- and pre-miRNA (Paper I; Meier et al., 2016) as in plants. However, our recent findings also suggest that the processing of pri-miRNAs involves at least one additional and so far unknown RNase (Paper I).

The developmental expression of many components of the RNAi machin-ery and the fact that also miRNAs are developmentally regulated (Avesson et al., 2012; Hinas et al., 2007; Paper I; Parikh et al., 2010), suggest that miRNAs play a role in the multicellular development of D. discoideum. However, so far no targets have been identified nor is it known which of the five Argonaute proteins that are dedicated to miRNA regulation.

Although much remains to be understood regarding miRNA biogenesis and Argonaute functions, published and ongoing research have started to clear the picture. Three of the Argonautes, A-C, have been shown to be involved in regulation of different transposable elements (Boesler et al., 2014; Malicki et al., 2020; Schmith et al., 2015). Furthermore, we have sequenced both mRNA and sRNA from three different Argonaute knock out strain, agnB, agnC, and agnE and compared their transcriptional profiles to WT cells and cells lacking miRNAs, i.e. DrnB knock out cells. Based on this, we detected that cells de-pleted of AgnB and DrnB were similarly affected both regarding mRNA reg-ulation and decrease of miRNA levels (Liao, Kjellin, Söderbom, un-published). Thus, our current knowledge suggests that AgnB is likely to be the miRNA associated Argonaute in D. discoideum.

So far, there are no direct evidence regarding how D. discoideum miRNAs find their targets or how the targets are regulated. Targets with full or near full complementarity should be easily predicted by computational analyses, but we have not been able to identify such targets (unpublished). Hence, we favor an animal-like target recognition mechanism, where the miRNA and the target mRNA interact with partial complementarity.

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Origin of miRNA regulation The last common ancestor of eukaryotes most likely had at least one Argo-naute and one Dicer. This ancestral RNAi machinery was probably involved in the defense against viruses and transposable elements via siRNA media si-lencing (Obbard et al., 2009). Subsequently, endogenous regulation with miR-NAs may have evolved from this system. However, when this occurred and how many times is still under debate.

As previously described, miRNA biogenesis and main mode of targeting differ between plants and animals (see above). Furthermore, miRNAs have mainly been identified in plants and animals and the overall sequence conser-vation of miRNAs are low (Moran et al., 2017). Combined, this has led to the assumption that miRNA regulation evolved independently multiple times.

There are also those who argue for a common ancestral miRNA regulatory mechanism, which evolved before the split of plants and animals. This is sup-ported by the increasing number of identified miRNAs in diverse organisms (Moran et al., 2017). The mode of miRNA targeting in the group of Cnidaria (e.g. sea anemones and jellyfish) have also been used as an argument for a common evolutionary origin of miRNAs. Phylogenetically, Cnidaria consti-tute a sister group to Bilateria within Metazoa (Moroz et al., 2014; Ryan et al., 2013). In contrast to bilaterian miRNAs, the miRNAs in cnidarians commonly regulate their target by cleavage via near-perfect complementarity similar to siRNAs and plant miRNAs (Moran et al., 2014). This suggests that targeting by seed complementarity evolved in bilaterians and that the common ancestral mode of regulation was cleavage through perfect complementarity (Moran et al., 2017). Taken together, these findings underscore the importance of inves-tigating miRNA regulation in other eukaryotic lineages than animals and plants. In addition, due to the high sequence variation of miRNAs, it will not be sufficient to identify miRNAs alone. Instead characterization of biogenesis and mode of targeting will be required.

The phylogenetic position of D. discoideum in Amoebozoa, in combination with its large toolbox for molecular studies, makes this an ideal system to im-prove our understanding of miRNA evolution. Our current knowledge of D. discoideum miRNAs indicate a more plant like system (see above), but there are also similarities to animal miRNAs. For example, similar to animals, miR-NAs appear not to be methylated during maturation as in plants (Avesson et al., 2012). Also, we suspect a more animal like miRNA targeting, i.e. by par-tial complementarity, but direct evidence is still needed. The determination of how miRNA regulates their targets in D. discoideum would be a major contri-bution to our understanding of their evolution. Based on the knowledge on miRNAs in other organisms, regulation via seed complementarity appears to have emerged in bilaterians (Moran et al., 2017). However, once we have ev-idence for the mode of targeting in D. discoideum, this model might need to be revised.

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Class I RNAs and multicellularity In addition to miRNAs, D. discoideum express another class of ncRNAs likely to be involved in multicellular development (Fig. 4a). Prior to the work pre-sented in this thesis, these Class I RNAs had only been identified in D. dis-coideum and D. purpureum which both belong to Group 4 of Dictyostelia (Aspegren et al., 2004; Sucgang et al., 2011). In D. discoideum, Class I RNAs are expressed from more than 30 genes and all share the potential to form a short stem connecting the 5’ and 3’ end (Fig. 4a) (Aspegren et al., 2004; Avesson et al., 2011). This structure has also been confirmed by chemical probing for one of the RNAs, DdR-21 (Avesson et al., 2011). Another strong feature of Class I RNAs, is a conserved 11 nt sequence motif, located adjacent to the 5’ part of the stem (Fig. 4a) (Aspegren et al., 2004; Eichinger et al., 2005).

Similar to many other ncRNAs in D. discoideum, Class I RNAs are pre-ceded by the putative promoter element DUSE (Aspegren et al., 2004; Hinas and Soederbom, 2007; Sucgang et al., 2011). Many Class I RNAs in D. dis-coideum also have a second upstream sequence motif, the TGTG-box, which so far only has been identified up-stream of Class I RNAs.

The function of Class I RNAs remains to be understood, but our current understanding suggests that they are involved in regulating multicellular de-velopment. Class I RNAs are abundantly expressed during vegetative growth, but at the onset of development their levels start to decrease (Aspegren et al., 2004; Avesson et al., 2011). Furthermore, cells depleted of one Class I RNA, DdR-21, are affected in their multicellular development (Avesson et al., 2011). This strain could still go through the entire multicellular development cycle but, compared to WT cells, more and smaller fruiting bodies were formed (Fig. 4b) (Avesson et al., 2011). This suggests that DdR-21 regulates early development and cause less cells to stream together to form smaller ag-gregates when absent. If Class I RNAs are regulating early development in D. discoideum, it may suggest that they are also present in other dictyostelids. The work presented in this thesis will address the spread of Class I RNAs in Dictyostelia and also other eukaryotic groups.

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Figure 4. D. discoideum Class I RNAs. a) Schematic representation of Class I RNAs, based on the sequence and structure of DdR-21. B) Developmental pheno-type of WT cells and cells depleted of DdR-21 (Avesson et al., 2011).

Host-pathogen interactions and gene regulation During a bacterial infection, phagocytic cells, e.g. macrophages, constitute the first line of defense (Medzhitov, 2007). These cells ingest the pathogen and attempt to degrade it in order to clear the infection. Some pathogens have evolved to avoid degradation and instead use the host cell for its own prolif-eration. In order to achieve this, the pathogen needs to manipulate the host immediately after uptake. This leads to a series of complex host-pathogen in-teractions which have been proven difficult to fully resolve. However, it is known that the transcriptome of the host cell is heavily affected upon infection by intracellular bacteria (Mogensen, 2009; Niller and Minarovits, 2016). This is partly due to the recognition of the bacteria by surface receptors of the host cell which activates defense systems that will try to degrade the pathogen after uptake (Cao, 2016). Once inside the host cell, the bacteria also actively ma-nipulate these defense systems in order to survive which in turn have a large impact on host gene regulation (Niller and Minarovits, 2016). Not surpris-ingly, regulatory ncRNAs are involved in this major rewiring of the transcrip-tome.

Both the ncRNAs of the host, e.g. miRNAs and lncRNAs, as well as bac-terial ncRNAs have been shown to shape the outcome of bacterial infections in mammalian cells (Duval et al., 2017). For example, part of the innate im-mune signaling triggered by surface receptors recognition of bacteria is medi-ated by miRNAs. In addition, several bacterial pathogens such as Helicobac-ter pylori, Salmonella spp. and Mycobacterium tuberculosis have been shown to actively manipulate the expression of a multitude of host miRNAs once inside the host (Duval et al., 2017).

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The recognition of pathogen motifs, e.g. lipopolysaccharides, by pattern recognition receptors (PRR) modulate the expression of at least three miR-NAs, miR-155, miR-146 and let-7, via the pro-inflammatory NF-κB pathway (Duval et al., 2017). High levels of NF-κB activity leads to increased expres-sion of pro-inflammatory factors via miR-155 regulatory pathways (Schulte et al., 2013) while low NF-κB activity activates miR-146 which function as an anti-inflammatory regulator (Schulte et al., 2013; Taganov et al., 2006). The let-7 family of miRNAs regulates multiple aspects of the innate immunity signaling (Duval et al., 2017). One of these, let-7f, targets an inhibitor of the NF-κB pathway and thereby increases its activity (Kumar et al., 2015). During infection, M. tuberculosis triggers a down-regulation of let-7f, which leads to increased bacterial survival in macrophages (Kumar et al., 2015).

Gene regulation during infection in D. discoideum The interaction between mycobacteria and the host cell during early infection has been extensively studied using the D. discoideum infection model (Car-denal-Muñoz et al., 2017b). However, prior to the work presented in this thesis very little was known about the mRNA regulation in the host upon infection and how representative this regulation is to human macrophages. In contrast, several studies have investigated the transcriptional response of D. discoideum to L. pneumophila by microarray analyses (Farbrother et al., 2006; Li et al., 2009). Notably, these microarrays did not cover all protein coding genes of D. discoideum. Compared to D. discoideum mRNA regulation upon infection, even less (i.e. nothing) was known about how ncRNAs were regulated during challenges with pathogenic bacteria.

With the work presented in this thesis, I have aimed to increase our under-standing of the regulation of host mRNA and ncRNA in response to intracel-lular bacterial pathogens.

Tuberculosis Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the dead-liest infectious diseases known and it is estimated that approximately one third of the worlds’ population carries a latent infection (Furin et al., 2019). TB infection is initiated by the inhalation of aerosols containing bacteria. Once the pathogen reaches the lower respiratory tract it is phagocytized by human immune cells, e.g. macrophages. However, M. tuberculosis evades degrada-tion and instead creates a replicative niche within the host cell (Schorey and Schlesinger, 2016). Eventually, the infected immune cells start to recruit other immune cells which leads to the formation of granulomas. These cellular ag-gregates form complex structures, which are still poorly understood but

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probably constitute a favorable environment for the bacteria (Pagán and Ra-makrishnan, 2014).

Today there is substantial evidence that Mycobacteria infection regulate miRNA levels in the host. In addition, they appear to actively manipulate the miRNA regulation in order to shape the host intracellular milieu in its favor (Agarwal et al., 2019).

Although nothing is known about the involvement of tRNA cleavage in the host during infection, M. tuberculosis is known to regulate its own growth via toxin-antitoxin (TA) systems in order to establish latent infections (Schifano and Woychik, 2017). This is achieved by the two toxins MazF and VapC, which cleaves specific tRNAs and generates tRNA-halves in the bacteria (Cruz et al., 2015; Schifano et al., 2016, 2013; Winther et al., 2016; Winther and Gerdes, 2011). Recently it was shown that the growth regulation induced after tRNA cleavage is due to ribosome stalling during translation, which leads to down-regulation of specific proteins (Barth et al., 2019).

M. marinum as a model for M. tuberculosis One of the closest relatives to Tuberculous mycobacteria, is the fish pathogen Mycobacterium marinum (Stinear et al., 2008). Although humans are not the primary host of this bacteria, it can cause granulomatous skin lesions. How-ever, due to its poor growth at 37 °C systemic spread is very rare in humans (Aubry et al., 2017). During experimental infections in fish, M. marinum causes an infection which is very similar to Tuberculosis, including the for-mation of granuloma and development into latent infection (Parikka et al., 2012). Furthermore, it shares several key virulence factors with M. tuberculo-sis such as five type 7 secretion systems, ESX1-5, which are required for the intracellular manipulation and survival within the host cell via the secretion of e.g. early secretory antigenic target (ESAT-6) protein (Stinear et al., 2008).

M. marinum infection in D. discoideum In 2003, Solomon and colleagues demonstrated that M. marinum can replicate within D. discoideum (Solomon et al., 2003). Later it was shown that the course of infection can be divided into three main stages, establishment of infection, bacterial replication, and arrested proliferation due to bacterial death or release (Hagedorn and Soldati, 2007).

Similar to the route of M. tuberculosis, M. marinum immediately manipu-lates the phagocytic pathway after uptake in D. discoideum to prevent phago-some maturation in order to create the mycobacteria containing vacuole (MCV) (Barisch et al., 2015; Cardenal-Muñoz et al., 2017a; Paper III). As a consequence, the vacuolar H+-ATPase (vATPase) as well as markers of late maturation, e.g. vacuolin and cathepsin D, are more or less absent from the MCV during early infection (Hagedorn and Soldati, 2007). Once the MCV

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has been established, several bacterial effectors are required for proliferation. One of these, MAG24-1 has been shown to be important for mycobacterial replication both within D. discoideum (Hagedorn and Soldati, 2007; Solomon et al., 2003) and macrophages (Ramakrishnan et al., 2000). In addition, the ESX-1 secretion system is also important for optimal bacterial replication (Cardenal-Muñoz et al., 2017a; Hagedorn et al., 2009) Later, M. marinum cause membrane damage to the MCV by ESAT-6 secretion via ESX-1, which facilitates its escape into the cytosol (Hagedorn et al., 2009; Hagedorn and Soldati, 2007). Both these roles of ESX-1 have also been observed in mam-malian cells (Gao et al., 2004; Simeone et al., 2015; Stamm et al., 2003; Tan et al., 2006; Volkman et al., 2004; Zhang et al., 2016).

Legionnaires disease The causative agent of Legionnaires disease is Legionella pneumophila, which was named after the first reported outbreak at a convention for members of the American Legion in 1976 (Fields et al., 2002). Infection in humans mainly occurs via inhalation of contaminated aerosols from human made wa-ter reservoirs, e.g. showers, which can lead to a severe type of pneumonia. Amoebae acts as natural reservoirs of the L. pneumophila, and can make the pathogen both more virulent and more resilient to disinfectants if present in the same water reservoir (Swart et al., 2018). However, in contrast to M. tu-berculosis, L. pneumophila infection in human normally constitute a dead-end for the bacteria. To my knowledge, there is only one reported case where trans-mission from person-to-persons probably occurred (Correia et al., 2016).

Infectious route of L. pneumophila The overall infectious route of L. pneumophila is very similar in D. dis-coideum and macrophages. In both hosts, the legionella containing vacuole (LCV) is formed during bacterial uptake. Within minutes, this membrane bound compartment evades transport to the lysosome and instead attracts mi-tochondria and endoplasmic reticulum (ER) derived vesicles. Eventually the ER vesicles covers the LCV and the enclosing membrane starts to resemble rough ER with ribosome distributed along the surface. L. pneumophila is now able to replicate to high numbers within the LCV which eventually will lead to lysis of the host cell (Isberg et al., 2009).

In both D. discoideum and macrophages, the formation of the LCV is very similar and is dependent on the Dot/Icm secretion system (Defect in Organelle Trafficking; Intracellular Multiplication) (Hägele et al., 2000; Segal et al., 1998; Segal and Shuman, 1999; Solomon et al., 2000; Vogel et al., 1998). With this type IV secretion system (T4SS), L. pneumophila translocate > 300 effector proteins into the host cell which act on diverse host cell pathways,

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such as signal transduction and membrane trafficking (Hubber and Roy, 2010). In macrophages and in the amoeba Acanthamoeba castellanii, Dot/Icm is also involved in enhancing the uptake into the host cell (Hilbi et al., 2001; Watarai et al., 2001). In macrophages, this has been shown to rely on the trans-fer of effector proteins into the host cell prior to uptake of L. pneumophila (Nagai et al., 2005).

So far, little is known about the involvement of the RNAi machinery in the host response to L. pneumophila infection. However, some studies provide evidence that L. pneumophila infection regulates specific miRNAs. These are mainly involved in regulating the inflammatory response in L. pneumophila infected cells, which can work both in favor of the pathogen or the host (Jentho et al., 2017; Jung et al., 2016; Koriyama et al., 2019). For example, L. pneu-mophila, and other gram-negative bacteria, can form outer membrane vesicles (OMVs). These can trigger an up-regulation of mir-146a leading to a down-regulation of interleukin-1 receptor-associated kinase 1 (IRAK1), resulting in increased bacterial replication (Jung et al., 2016). Recently, a more large-scale characterization of the host miRNA response to L. pneumophila infection showed differential regulation of 85 miRNAs (Herkt et al., 2020). Three of these were shown to act cooperatively to regulate host immune response which in turn restricted L. pneumophila growth.

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Current investigations

Aim The overall aim of this thesis is to gain further knowledge on the evolutionary origin of two types of ncRNAs, i.e. Class I RNAs and miRNAs. In addition, I have aimed to provide an increased understanding of the host response to in-tracellular bacteria by investigating the transcriptional response, including ncRNA, after infection. These studies have their starting point in dictyostelid social amoebae, and in particular D. discoideum, but the results are also put into the context of eukaryotic evolution.

First, we investigated the role of the Dicer-like protein DrnB in miRNA biogenesis in D. discoideum and characterized the transcription and pro-cessing of two pri-miRNAs (Paper I). Next, we studied the evolution of Class I RNAs and further investigated their involvement in dictyostelid aggregative multicellularity (Paper II). Finally, we characterized the host regulation of both mRNAs and small (s)RNAs in response to intracellular bacterial infec-tion using the D. discoideum infection model and compared it to responses induced in macrophages (Papers III and IV)

Biogenesis of miRNAs in D. discoideum (Paper I) The RNAi machinery is ancient and was most likely present in the last com-mon ancestor of animals and plants. However, if this ancestral RNAi machin-ery regulated genes with miRNAs, or if it was restricted to siRNA silencing, is still under debate. In order to improve our understanding of miRNA evolu-tion, more information is needed regarding the presence and function of miRNA regulation in other eukaryotic lineages than plants and animals.

DrnB is essential for biogenesis of all miRNAs We have previously shown that the Dicer-like protein DrnB is required for the biogenesis of four miRNAs in D. discoideum (Avesson et al., 2012; Hinas et al., 2007). In order to further investigate the role of DrnB in the cell, we per-formed sRNA-seq of both wild-type (WT) and drnB knock out cells during unicellular growth and the multicellular slug stage.

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We showed that depletion of DrnB have a limited effect on the overall sRNA population (18-40 nt) in the cell. In both WT and drnB k.o. strains, the majority of the sRNAs were 21 nt long and no major changes were detected in the levels of sRNAs derived from different classes of RNA, e.g. mRNA and various ncRNAs. However, when we focused on mature miRNAs we detected strongly reduced levels in cells depleted of DrnB.

Next, we analyzed the RNA-seq data from WT cells and identified 10 new miRNAs. Most of these fulfilled stringent criteria for miRNA annotation in plants and animals (Kozomara and Griffiths-Jones, 2014). In addition, the ma-jority of the new miRNAs were developmentally regulated and all were strongly dependent on DrnB for correct processing.

Taken together, these results indicate that DrnB is essential for global miRNA biogenesis while it is dispensable for the generation of most other sRNAs in the cell.

Precursor transcript of miRNA are stabilized in DrnB depleted cells The lack of mature miRNAs in drnB k.o. strain indicated that the stem loop is not processed and therefore might be stabilized in these cells. In order to in-vestigate this, we performed RNA-seq on poly(A) enriched RNA from the same life stages as used for the sRNA-seq. Interestingly, we found reads cov-ering both the predicted stem loop but also extending into the up-stream and downs-stream regions. This suggested that we could detect longer miRNA precursor transcripts, here on referred to as pri-miRNAs, by RNA-seq. Fur-thermore, differential expression analysis revealed that most of the pri-miR-NAs are stabilized in the absence of DrnB both during unicellular growth and multicellular development. This was also confirmed for a subset of the pri-miRNAs by semi-quantitative RT-PCR.

Biogenesis of miRNA-1176 and miRNA-1177 The elevated levels of pri-miRNAs in the absence of DrnB allowed us to study the biogenesis of miRNAs in more detail. For this we focused on two of the most abundant miRNAs in D. discoideum, i.e. mir-1176 and mir-1177, and investigated the 5’ and 3’ ends of both pri-miRNAs and processing interme-diates.

Determination of the 5’ end gave similar result for both pri-mir-1176 and pri-mir-1177, where both transcripts were shown to start with G residue close to the 5’ most read in the RNA-seq data. In contrast, characterization of the 3’ end revealed differences in the processing of the two pri-miRNAs. For pri-mir-1176, we identified both full-length pri-miRNAs and processing interme-diates. Interestingly, the full length pri-miRNAs was identified in both WT

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and drnB k.o. strains while the processing intermediates were found to be sta-bilized in the absence of DrnB. This suggests that pri-mir-1176 is cleaved by another, so far unknown, RNase prior to DrnB processing. To our surprise, we found that the full-length pri-mir-1176 was not polyadenylated while the shorter processing intermediates, stabilized in the drnB k.o. strain, carried short post-transcriptionally added A-tails. The function for these short A-tails remains to be elucidated.

We were not able to determine the 3’ end of the full length pri-mir-1177. However, we show that in the absence of DrnB, transcription of pri-mir-1177 extends into the gene situated downstream of the mir-1177 locus. We con-firmed that the downstream gene is transcribed from its own promoter and that its expression appears less affected by DrnB depletion than pri-mir-1177. This suggests that pri-mir-1177 is independently transcribed and is not part of the 5’ UTR of the downstream gene.

In conclusion, we demonstrated in this study that DrnB is essential for the correct processing of all identified miRNAs in D. discoideum. Furthermore, it appears to be dedicated to this task since limited effects were detected on other sRNAs in cells depleted of DrnB. Finally, we showed that precursor tran-scripts are stabilized in the absence of DrnB, which allowed detailed charac-terization of both miRNA gene transcription and processing of precursor tran-scripts.

Evolution of Class I RNAs and multicellular development (Paper II) We previously identified and characterized a novel type of non-coding RNA called Class I RNAs. Our previous investigations indicate a role of Class I RNAs in the multicellular development of D. discoideum (Aspegren et al., 2004; Avesson et al., 2011). In this study, we further explored the involvement of Class I RNAs in dictyostelid multicellularity by investigating their conser-vation both within and outside of Dictyostelia.

Prediction of Class I RNAs in evolutionary distant organisms The overall sequence similarity of previously identified Class I RNAs is low even within the same organism. Hence, reliable prediction of Class I RNA genes in evolutionary distant organisms cannot be performed by purely se-quence based approaches e.g. BLAST. Therefore, we created a co-variance model (CM) based on both sequence and consensus structure of 34 previously identified D. discoideum Class I RNAs.

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First, we searched well-studied dictyostelids representing each major group of Dictyostelia, i.e. D. fasciculatum (Group 1), P. pallidum and A. subglo-bosum (Group 2A and B), D. lacteum (Group 3) and D. purpureum and D. discoideum (Group 4). Interestingly, Class I RNA gene candidates were iden-tified in all organisms and the sequence and structure information of these were used to create a refined version of the CM. With the new model we could identify more candidate genes which were in turn added to the CM. This pro-cess was repeated until no new candidates were found. In the end, we could identify 126 Class I RNA candidate genes distributed over all groups of Dic-tyostelia.

Construction of a Class I RNA classifier In both D. discoideum and D. purpureum, Class I RNA genes are preceded by a putative promotor element called DUSE (Dictyostelium upstream element) (Aspegren et al., 2004; Sucgang et al., 2011). In addition, many D. discoideum Class I RNA genes also have a second up-stream sequence motif, the TGTG-box. Interestingly, we found that DUSE and its distance from the transcrip-tional start site are conserved throughout Dictyostelia while the TGTG-box was only identified in D. discoideum. We used this information to create a classifier, which evaluates the CM search result based on the presence and location of these sequence motifs. Each candidate was scored based on the presence of DUSE at the expected upstream location. Absence of DUSE or non-canonical distance was penalized with negative score. The sequence mo-tif and distance scores were combined with the score obtained in the CM search and a classifier score threshold was set to allow identification of diver-gent Class I RNAs. Using this approach, we predicted 18-39 Class I RNA genes per organism of which the majority had DUSE at the expected up-stream location.

Class I RNAs are expressed in all groups of Dictyostelia The high number of predicted genes per organism suggested that Class I RNAs are conserved in all major groups of Dictyostelia. However, previous to this study, Class I RNA expression had only been confirmed in D. discoideum (Aspegren et al., 2004; Avesson et al., 2011). In order to test if they are ex-pressed also in the other dictyostelids, we performed northern blot analyses of Class I RNA candidates in representatives of all four groups of Dictyostelia. We confirmed the expression of all tested candidates but also found that sev-eral Class I RNAs were slightly larger than predicted.

In order to gain a more complete picture of Class I RNA expression, we performed RNA-seq on both growing and developed cells of D. fasciculatum (Group 1), P. pallidum (Group 2), D. lacteum (Group 3) and D. discoideum (Group 4). Based on RNA-seq read count and coverage, we could prove that

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the majority of the predicted Class I RNAs are indeed expressed. Furthermore, we detected that the start of many mature Class I RNAs were 1-2 nucleotides earlier than predicted, which is in line with the slightly larger sizes detected by northern blot analyses. Based on the expression validation we could also evaluate the performance of our classifier which proved to be both highly ac-curate and sensitive in predicting Class I RNAs in all groups of Dictyostelia.

Taken together, northern blot and RNA-seq analyses proved that Class I RNAs are present in all investigated dictyostelids and expressed at high levels. In addition, we showed that the Class I classifier can reliably predict expressed Class I RNAs in evolutionary distant dictyostelids with almost no false posi-tives.

Key features of D. discoideum Class I RNAs are highly conserved throughout Dictyostelia Comparison of all the identified Class I RNA loci revealed several strongly conserved features. Transcription appears to be dependent on DUSE as all ex-pressed Class I RNA genes have a DUSE at the correct up-stream location. Furthermore, we detected stretches of thymine residues downstream of the majority of Class I RNA loci, which likely constitute a conserved terminator for RNA polymerase III transcription (Richard and Manley, 2009). The ma-ture transcripts all have the potential to form a short stem structure connecting the 5’ and 3’ end where the 5’ terminal G residue of the stem is perfectly conserved (Fig. 4). Furthermore, the 11 nt sequence motif found adjacent to 5’ part of the stem in D. discoideum and D. purpureum is present in all iden-tified Class I RNAs. Some nucleotides in the 11 nt sequence motif have been allowed to vary during Class I RNA evolution while others are strongly con-served.

Although the function of Class I RNAs remains to be understood, the con-servation of the sequence motif and stem structure suggest that these are key functional features. We observed low sequence conservation of the stem be-tween species. In addition, a high number of compensatory mutations within each species have led to a varying sequence of the stem while the structure has been kept. Taken together, this suggests that it is the stem structure and not sequence that is important for Class I RNA function.

Class I RNAs are developmentally regulated and ubiquitous in Dictyostelia We have previously shown that the expression of Class I RNAs are develop-mentally regulated in D. discoideum (Group 4) and that the disruption of DdR-21 cause aberrant multicellular development (Aspegren et al., 2004; Avesson et al., 2011). RNA-seq analyses demonstrated that Class I RNAs in Group 1-

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2 species are also developmentally regulated. This led us to hypothesize that Class I RNAs are involved in the multicellular development of all members of Dictyostelia.

Next, we used the classifier to identify the presence of Class I RNAs in ten additional dictyostelids. In line with our hypothesis, we identified 9-31 Class I RNA genes in each genome of which the majority also fulfilled the require-ments for being expressed, i.e. presence of DUSE at expected upstream loca-tion. In addition, further characterization revealed that the previously identi-fied key features of Class I RNAs were still strongly conserved in all identified loci. The TGTG-box, previously identified only in D. discoideum, was found in four additional species all belonging to Group 4 or the closely related P. violaceum complex.

Taken together, the presence of Class I RNA genes in all tested dictyoste-lids strongly suggest that Class I RNAs were present in the LCA of Dictyoste-lia.

The emergence of Class I RNAs correspond to the evolution of multicellular development in Dictyostelia To further investigate the spread of Class I RNAs, we searched genome se-quences of organisms outside of Dictyostelia. Outgroups where chosen to rep-resent the closest unicellular relatives within Amoebozoa as well as organisms representing other major eukaryotic groups. In addition, we included the pro-teobacterium Myxococcus xanthus as it, similar to dictyostelids, exhibits ag-gregative multicellularity upon starvation (Muñoz-Dorado et al., 2016). Inter-estingly, no Class I RNA genes could be identified outside Dictyostelia sug-gesting that this type of ncRNA is restricted to dictyostelid amoebae. The re-sults indicate that the emergence of Class I RNAs coincide with the LCA of Dictyostelia.

Conservation of interacting proteins We know from our previous work that one D. discoideum Class I RNA, DdR-21, associates with four different proteins. One of these, the RNA recognition motif containing protein CIBP, was shown to directly interact with the RNA (Avesson et al., 2011). We investigated the conservation of this protein and found that is likely to be present in all members of Dictyostelia. The CIBP mRNA was also shown to be similarly regulated during development in rep-resentatives of each group of Dictyostelia. Interestingly, this regulation resem-bles the changes in Class I RNA levels, including DdR-21, during early de-velopment.

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In conclusion, in this study we developed a classifier capable of sensitive and accurate prediction of expressed Class I RNAs in evolutionary distant dicty-ostelids. By using this approach, we show that Class I RNAs are conserved throughout Dictyostelia and was therefore most likely present in the last com-mon ancestor of this evolutionary distinct group. Several remarkably well con-served features were identified, suggesting that they are important for Class I RNA function.

Host mRNA response to infection by intracellular bacteria (Paper III) The D. discoideum infection model have successfully been used to elucidate many aspects of the interactions between intracellular bacterial pathogens and the host. These host-pathogen interactions have a major impact on the host transcriptome in mammalian immune cells (Mogensen, 2009; Niller and Minarovits, 2016). However, little is known about the mRNA regulation in D. discoideum upon infection and if it is representative for the transcriptional re-sponses in macrophages. In this study, we use the D. discoideum infection model to investigate the mRNA regulation of the host during early infection by M. marinum and L. pneumophila.

Both M. marinum and L. pneumophila infection have a strong impact on host mRNA levels shortly after uptake To investigate the transcriptome of D. discoideum during early infection by M. marinum and L. pneumophila respectively, we sequenced poly(A) selected RNA from infected and non-infected cells. Differential expression analysis identified 440 genes regulated in response to M. marinum 2.5 hours post in-fection and 330 and 1300 regulated in response to L. pneumophila at 1 and 6 h post infection, respectively. The regulation of a subset of 12 genes, repre-senting both up- and down-regulated as well as non-regulated after M. mari-num infection, were confirmed by qPCR using the same RNA as for the RNA-seq. In addition, we performed new infection experiments and proved that the regulation of these genes was highly reproducible both considering direction and level of regulation.

Distinct host responses detected after M. marinum and L. pneumophila infection In order to gain a better understanding of the transcriptional responses, we performed gene ontology (GO) enrichment analyses as well as compared our results to published literature. We identified many regulated genes in response

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to both M. marinum and L. pneumophila which previously have been shown to be part of the host response to these pathogens in both amoebae and mac-rophages. In addition, we could identify large sets of genes that have previ-ously not been characterized in association with bacterial infection.

M. marinum infection induced genes associated with small GTPase signal-ing, the endosomal sorting complex required for transport (ESCRT) machin-ery and autophagy. Only a small subset of the regulated genes was found to be down regulated (9 %). Among these we mainly observed an enrichment of transporter genes involved in e.g. metal ion transmembrane transport. Taken together, our results indicated that M. marinum induce host mRNA regulation which likely affects phagosome maturation, intracellular trafficking and au-tophagy.

The response to L. pneumophila infection was overall very different com-pared to the regulation detected in cells infected with M. marinum. Among the up-regulated genes, we observed an enrichment for tRNA-metabolism genes and genes involved in reactive oxygen species (ROS) production and scav-enging. In contrast to M. marinum infected cells, we observed a large set of strongly repressed cellular processes in response to L. pneumophila infection. Many important ribosomal biogenesis factors, such as the PeBoW complex, was down-regulated. In addition, rRNA transcription is potentially impaired due to a down regulation of RNA Pol I complex genes. At the later timepoint, 6 hpi, we also detect a down regulation of genes involved in cellular primary metabolism and energy production, e.g. pyruvate dehydrogenase complex and ATP citrate synthase genes. This suggests that L. pneumophila infection have a major impact on the host translational machinery and energy production.

Comparison of host responses reveals potential common defense genes The regulated processes identified by GO-term analyses after M. marinum and L. pneumophila infections show that the two pathogens trigger distinct host responses. However, when we compared the responses on gene level, we found a large set of genes that were similarly regulated in response to both M. marinum and L. pneumophila. This shared response involved several small GTPases as well as iron transporters and RNAi associated genes. Interestingly, this shared infection induced response in D. discoideum was distinct from the one triggered under normal growth when the amoeba utilizes Escherichia coli as food bacteria. In summary, this indicates that there is a common regulatory response in the host during bacterial infection.

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Similar regulation identified in infected macrophages Finally, we wanted to investigate if the regulation detected in D. discoideum upon bacterial challenge is representative for the response in macrophages af-ter infection. Therefore, we compared the D. discoideum transcriptional re-sponse to the regulation in human monocyte-derived macrophages infected with M. tuberculosis or L. pneumophila (Blischak et al., 2015; Price and Abu Kwaik, 2014). Despite the large evolutionary distances between the hosts as well as different mycobacteria used for the infection, similar regulation were found for many key genes. For example, an up-regulation of ESCRT compo-nents and down-regulation of PeBoW complex genes was detected in both amoebae and macrophages in response to M. tuberculosis and L. pneumophila respectively. In addition, an up-regulation of RNAi associated genes, e.g. Ago2, was found in infected macrophages which resembles the common reg-ulation to M. marinum and L. pneumophila in D. discoideum.

In conclusion, our study shows that the two pathogens M. marinum and L. pneumophila trigger distinct mRNA regulation in D. discoideum. Both patho-gens also induce a common response which differs from the response induced by feeding on bacteria. Furthermore, we show that many key genes are simi-larly regulated in human macrophages after infection, thus improving the va-lidity of the D. discoideum infection model.

Infection by mycobacteria trigger cleavage of specific host tRNAs (Paper IV) In Paper III we found a potential role of the RNAi machinery in the host re-sponse to infection. Therefore, we also wanted to investigate the effects on the sRNA population of the host during intracellular bacterial infection. By now, it is well established that host ncRNAs, e.g. miRNAs, play an important role in the host-pathogen interactions in mammalian macrophages (Duval et al., 2017). However, so far no one has investigated how the ncRNome of D. dis-coideum is affected during infection.

M. marinum causes specific tRNA cleavage in the host In order to investigate the effect of intracellular bacterial infection on the small RNA repertoire of D. discoideum, we performed sRNA-seq using the same RNA-samples as in Paper III. In non-infected and L. pneumophila infected cells, size distribution revealed that the majority of the sequenced sRNAs were 20-22 nt long while an even distribution was seen over the other sizes. This is in line with previous sRNA sequencing of growing and developed D. dis-coideum cells (Paper I; Avesson et al., 2012). In contrast, M. marinum

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infection caused a major shift in the sRNA size distribution and a strong en-richment of 33 nt long sRNAs was observed (43 % compared to 1-3 % in the L. pneumophila infected and non-infected cells).

These 33 nt RNAs correspond to the 5’ tRNA half of tRNA-Aspartate-GUC and constitute more than 35 % of all sequenced sRNAs in M. marinum infected samples. Furthermore, we show that these tRNA halves are generated also in non-infected and L. pneumophila infected cells although at much lower levels (≤ 1 %). This suggest that the tRNA cleavage is performed by an en-dogenous cleavage pathway. Interestingly, no sRNA corresponding to the 3’ half could be identified in the RNA-seq data. Taken together, these results suggest that M. marinum cause a dramatic and specific increase of 5’ tRNA-Asp-GUC halves in infected cells compared to non-infected and L. pneumoph-ila infected cells.

The tRNA-response to M. marinum is not restricted to the 5’ halves of tRNA-Asp In order to further investigate the effect on tRNA-Asp during infection, we performed northern blot analysis on RNA from M. marinum and L. pneumoph-ila infected D. discoideum cells. We also included RNA from amoebae sub-jected to Klebsiella pneumonia, commonly used as food for D. discoideum. This analysis confirmed the increase of 5’ tRNA-Asp halves in response to M. marinum infection. Low levels of 5’ tRNA-Asp-halves were also detected in non-infected cells as well as cells challenged with L. pneumophila or K. pneu-monia. These results support that the halves are generated by an endogenous cleavage pathway of D. discoideum which is induced during M. marinum in-fection.

To our surprise, we also detected high levels of 3’ tRNA-halves in M. mari-num infected cells despite the complete lack of these in RNA-seq data. North-ern blot analyses also revealed that the tRNA halves generated in M. marinum infected cells are slightly larger than the ones detected in other conditions. Perhaps M. marinum induce extra modifications of tRNAs which leads to their degradation?

The RNA samples from infected cells used for RNA-seq and northern blot analyses were isolated from a mixture of amoebae which had taken up the pathogen and those that had not. Therefore, we cannot conclude based on this data if the bacteria actually need to get inside the host cell in order to induce this response. In order to gain some insight into this, we infected D. dis-coideum cells with GFP-expressing M. marinum and sorted the cells which had intracellular bacteria (GFP-positive) from the once that had not (GFP-negative). Interestingly, similar levels of 5’ tRNA-Asp-halves were detected in both infected and non-infected cells. This indicates that either the bacteria

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or the host secretes an unknown factor which induce this response, regardless if M. marinum is taken up by the host cell or not.

In summary, northern blot analyses confirmed most of the RNA-seq results but also revealed a much more complex tRNA response to M. marinum infec-tion involving both halves of tRNA-Asp which appears to be generated from modified tRNA molecules. These results also showed that RNA cleavage dy-namics cannot be reliably studied by standard sRNA-seq methods alone.

Host tRNA cleavage is a conserved response to mycobacterial infection In recent years, tRNA-halves and shorter tRNA fragments have received a lot of attention and have been associated with a wide range of biological pro-cesses (Shen et al., 2018). However, we were unable to find a previously re-ported connection between tRNA cleavage and host response to bacterial in-fection. In order to investigate if the dramatic effect on the tRNA population in D. discoideum is representative for infected macrophages, we re-analyzed previously published data from macrophages infected with different mycobac-terial species and L. pneumophilia (Furuse et al., 2014). Based on this, we showed that a similar response occurs in mammalian macrophage cell lines (RAW264.7 and THP-1) during infection. High levels of specific 5’ tRNA halves, i.e. halves from tRNA-Valine and tRNA-Glycine, were found in re-sponse to mycobacteria, including M. tuberculosis, but not L. pneumophila.

In conclusion, infection by mycobacteria induce major changes in the small RNA population of D. discoideum characterized by a dramatic enrichment of tRNA-Asp halves. This response is specific to mycobacteria as it was not trig-gered by L. pneumophila infection or feeding on K. pneumonia. Furthermore, a similar response is triggered in mammalian macrophages during mycobac-terial infection.

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Concluding remarks and future perspectives

The findings presented in this thesis do not constitute an end but rather a start-ing point for further studies. I hope, and believe, that my work has contributed to our common knowledge about the evolution of miRNAs and Class I RNAs and their potential involvement in multicellularity. I have also provided new findings on the transcriptional response, including ncRNAs, of the host during infection by intracellular bacterial pathogens. Intriguingly, these responses ap-pear, in many aspects, to be conserved between amoebae and macrophages. However, as always, many questions remain to be answered.

Evolution of miRNAs and their connection to multicellularity In Paper I, we studied the biogenesis of miRNAs in D. discoideum. Investiga-tions such as this have the potential to increase our understanding about the miRNA evolution. In order to further study the origin of miRNA regulation, we want to investigate the presence of miRNAs throughout Dictyostelia.

Similar to Paper III where we studied the evolution of Class I RNAs, we want to investigate the presence (and appearance) of miRNA in social amoe-bae representing each major group of Dictyostelia. In addition, we will include non-dictyostelid amoebae to understand if miRNAs also are present in unicel-lular amoebozoans. This has the potential to greatly increase our understand-ing regarding the emergence of miRNA regulation in Dictyostelia and also in eukaryotes in general. Furthermore, this set up will allow us to investigate the correlation between increased organismal complexity and the number of (dif-ferent) miRNAs. This correlation can be seen in animals where miRNAs are believed to have been fundamental for the evolution of multicellularity. Whether this holds true also for the different groups within Dictyostelia re-mains to be understood.

This project is a major undertaking since miRNAs cannot be reliably de-tected based on genome sequence alone but require small RNA sequencing of each species. Furthermore, our knowledge about miRNAs in D. discoideum suggests that sequencing needs to be performed at a great depth also in other dictyostelids to detect miRNAs in the wealth of siRNA. In order to increase the chance of detecting developmentally regulated miRNAs, RNA from both vegetative growing cells as well as several stages during multicellular devel-opment should preferably be sequenced.

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Another important question is how miRNA binds to their target (m)RNAs in Dictyostelia. Our current knowledge on miRNA targeting in other organ-isms suggests that miRNA regulation via seed complementarity emerged in bilaterians (Moran et al., 2017). However, since no near-perfect miRNA target sites have been identified in D. discoideum, we believe that miRNA regulation is executed by partial complementarity. In order to investigate this further, we plan to use an RNA-seq approach where we investigate the mRNA regulation in cells lacking miRNAs (drnB k.o.) with the regulation in strains over ex-pressing single miRNAs. Based on this data, we aim to find target candidates by looking for mRNAs that are up-regulated in the absence of miRNAs and down-regulated when a miRNA is over expressed. These candidates can then be further analyzed for potential target sites which in turn can be validated experimentally.

Class I RNA function In Paper II, we demonstrated that Class I RNAs are conserved in all dictyoste-lids but are likely to be absent in other eukaryotes. These results together with previous work, suggest that Class I RNAs emerged around the same time as dictyostelid multicellularity. However, we still do not know where they orig-inate from. Previous studies have shown that several factors essential for dic-tyostelid development are likely to have been acquired through horizontal gene transfer (Glöckner et al., 2016). Can the same be true for Class I RNAs? This could in principal be investigated with the co-variance model we created for Class I RNAs in Paper II.

So far, no direct function has been demonstrated for these ncRNAs. How-ever, we have recently started a project where we attempt to elucidate the function of one D. discoideum Class I RNA, DdR-21. This Class I RNA was previously shown to affect early development and to directly interact with at least one protein, CIBP. In Paper II, we show that this protein is also conserved throughout Dictyostelia and share expression pattern with DdR-21 during early development. Unfortunately, any attempts to produce a CIBP k.o. strain have so far been unsuccessful. Instead, we are hoping to gain insight into the function of DdR-21 by combining RNA-seq with proteomics and compare the mRNA and protein regulation in WT cells and DdR-21 k.o. cells. We will sample both strains during vegetative growth as well as regular intervals dur-ing early multicellular development. We anticipate that this approach will de-tect differences in the DdR-21 k.o. cells compared to WT cells which can help us elucidate the function of this Class I RNA.

Host-pathogen interaction and the non-coding RNome In Paper III and IV, we characterized the host response to intracellular bacte-rial infection on both mRNA and sRNA level by RNA-seq. We demonstrate

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that the two pathogens, M. marinum and L. pneumophila, induce distinct re-sponses that also in many ways reflect the regulation in macrophages after infection. This conservation is remarkable considering the vast evolutionary distance between the two hosts, but also suggests that the detected processes are highly relevant to understand the host-pathogen interaction.

We have demonstrated that mycobacteria infection trigger cleavage of spe-cific host tRNAs in both amoebae and macrophages. However, what enzyme is generating these fragments? From other organisms, we know that tRNA halves can be generated by Angiogenin and various RNase T2 family proteins (Gebetsberger and Polacek, 2013). No Angiogenin homologue have been identified in D. discoideum but genes encoding RNase T2 proteins have been identified. Therefore, we have ordered knock out strains of several RNase T2 genes which we plan to infect with M. marinum. If we do not detect an increase of tRNA halves after M. marinum infection in one of these mutant strains, it will be a good indication that we have identified the enzyme responsible for tRNA cleavage in D. discoideum. If this succeeds, it will be highly interesting to see if the lack of tRNA halves during M. marinum infection will be benefi-cial for the host or the bacteria.

Our results in Paper IV also suggested that tRNA cleavage might be in-duced by a secreted factor by either the bacteria or host cells. In order to gain further insight into this, we plan to subject D. discoideum cells to supernatant from M. marinum cultures as well as from infected D. discoideum cultures to see if we can trigger tRNA cleavage.

Finally, we would like to further investigate the RNAi response and in-volvement of D. discoideum miRNAs during infection. Initial analyses indi-cate that several miRNAs are induced during L. pneumophila infection while no clear involvement of miRNA regulation was detected in response to M. marinum. However, this needs to be further investigated as the high proportion tRNA fragments in the sRNA libraries from M. marinum infected cells may conceal what is happening in the population of smaller RNAs.

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Svensk sammanfattning

En människa består av många miljarder celler varav alla har samma genupp-sättning. Dessutom är många av dessa gener och de proteiner som de kodar för förvånansvärt lika de som finns i t.ex. jästsvampar och amöbor. Hur är detta möjligt? Svaret ligger till stor del i förmågan att reglera vilken gen som uttrycks, när den uttrycks och hur starkt detta genuttryck ska vara. Förmågan att reglera genuttryck är dessutom livsviktig för cellers förmåga att snabbt an-passa sig till förändrande förhållanden, som t.ex. svält eller infektion.

Idag vet vi att denna genreglering till stor del är beroende av icke-kodande RNA. Begreppet icke-kodande hänvisar till att dessa RNA inte translateras till proteiner utan istället utför olika funktioner som just RNA molekyler. Speci-ellt en klass av icke-kodande RNA, mikroRNA, har visat sig vara viktig i en mängd olika processer. Trots att de bara är ungefär 21 nukleotider långa så beräknas de reglera uttrycket av fler än hälften av våra gener. Dessa mikroRNA finns i de flesta djur och växter men har än så länge bara identifi-erats i några få andra typer av organismer.

En av dessa är den sociala amöban Dictyostelium discoideum som tillhör den evolutionära gruppen Dictyostelia. Alla amöbor som tillhör Dictyostelia lever som encelliga organismer så länge som det finns bakterier eller andra mikroorganismer som de kan äta. Men om maten tar slut så strömmar amö-borna samman i klumpar som kan innehålla över 100 000 celler. Amöborna börjar nu samarbeta som en flercellig organism där vissa celler till slut bildar sporer medan andra offrar sig för att bilda en stjälk av döda celler. Amöborna som har bildat sporer samlas i en boll högst upp på stjälken där de sedan kan spridas vidare med vinden för att förhoppningsvis landa på en ny plats där det finns mat.

D. discoideum är en väletablerad modellorganism som har använts för att studera en mängd olika biologiska processer. Dessutom har den använts som modell för mänskliga immunceller som t.ex. makrofager. Både makrofager och D. discoideum tar upp mikroorganismer genom en process som kallas fagocytos för att sedan bryta ner dessa genom konserverade intracellulära pro-cesser.

Förekomsten av mikroRNA i D. discoideum är intressant av flera anled-ningar. Dels så kan studier av mikroRNA i denna organism utöka vår förstå-else för hur genreglering med hjälp av mikroRNA uppstod under evolutionen. Dessutom så är dessa icke-kodande RNA starkt förknippade med flercellighet hos djur. Om mikroRNA är inblandade i regleringen av flercellighet också hos

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sociala amöbor återstår att se. Dock så vet vi att en annan klass av icke-ko-dande RNA är inblandade i att reglera deras multicellulära utveckling. Dessa icke-kodande RNA kallas Class I RNA och har tidigare bara upptäckts i D. discoideum och dess nära släkting D. purpureum.

Med de studier som presenteras i denna avhandling, har vi med hjälp av D. discoideum och andra sociala amöbor undersökt flera aspekter av genreglering och evolution av icke-kodande RNA. I Artikel I, så undersökte vi hur mikroRNA bildas i D. discoideum. Genom att slå ut genen som kodar för pro-teinet DrnB visade vi att det är oumbärligt för produktion av mikroRNA. Sam-tidigt så ökade nivåerna av prekursorerna, det vill säga de längre RNA-mole-kylerna från vilken mikroRNA bildas, vilket medförde att vi kunde studera dessa i detalj. Våra resultat visar att de processer som leder till bildandet av mikroRNA i D. discoideum på många sätt påminner om dessa processer hos växter. Sammanfattningsvis kan dessa resultat bidra till vår förståelse för när under evolutionen som genreglering med hjälp av mikroRNA uppstod.

I Artikel II, undersöker vi evolutionen av Class I RNA och dess koppling till flercellighet. Vi utvecklade en sökmetod för att hitta gener för Class I RNA i organismers genomsekvenser. Vi kunde sedan visa att denna metod identifi-erar uttryckta Class I RNA gener i evolutionärt avlägsna organismer med stor träffsäkerhet. Baserat på detta kunde vi visa att Class I RNA finns i alla sociala amöbor som hör till gruppen Dictyostelia, medan de närmst besläktade encel-liga amöborna saknar denna klass av icke-kodande RNA. Sammantaget indi-kerar vår studie att uppkomsten av Class I RNA sammanfaller med uppkoms-ten av flercellighet hos sociala amöbor.

I Artikel III och IV så utnyttjar vi likheten mellan amöbor och mänskliga immunceller för att undersöka hur värdcellens RNA-nivåer påverkas vid in-fektion av intracellulära patogena bakterier. Dessa bakterier tas upp av både immunceller och amöbor, men istället för att brytas ner så undviker bakteri-erna värdcellens försvarsmekanismer. Genom att manipulera värdcellen på-verkar bakterien den intracellulära miljön för sina egna ändamål.

Tidigare studier har visat att det finns stora likheter i interaktionen mellan värdcell och bakterie vid infektion i amöbor och mänskliga immunceller. I Artikel III och IV visar vi att detta också stämmer när det gäller värdcellens reglering av RNA-nivåer vid infektion av Mycobacterium marinum respektive Legionella pneumophila. Artikel III fokuserar på regleringen av budbärar-RNA, dvs de RNA-molekyler som translateras till proteiner. Genom att stu-dera regleringen av alla kända budbärar-RNA i infekterade och icke-infekte-rade celler så kunde vi se att de två patogena bakterierna till största del påver-kar uttrycket av olika gener under infektion. Vi visar också att många besläk-tade gener i mänskliga celler påverkas på samma sätt vid infektion av Myco-bacterium tuberculosis och L. pneumophila.

Trots att majoriteten av den regleringen som vi detekterade i D. discoideum visade sig vara unik för respektive bakterieinfektion så fanns det ett antal ge-ner som påverkades på samma sätt. När vi studerade funktionen hos dessa

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gener så såg vi att gener för proteiner inblandade i genreglering via mikroRNA var påverkade. Detta ledde till artikel IV där vi undersöker om denna reglering också reflekterade en påverkan på cellens små RNA molekyler. Vi upptäckte att M. marinum infektion hade en stor påverkan på en annan typ av icke-ko-dande RNA, nämligen tRNA. Våra resultat visade att infektion av M. marinum orsakade en drastisk ökning av specifika tRNA-molekyler som kluvits itu i värdcellen. När vi undersökte data från infekterade makrofager kunde vi se att detta sker även i dessa celler efter infektion av M. tuberculosis.

Sammanfattningsvis så har studierna i denna avhandling ökat vår förståelse för evolutionen av två klasser av icke-kodande RNA samt bidragit till vår kun-skap om de komplexa interaktioner som sker mellan intracellulära bakterier och deras värdcell.

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Acknowledgements

First of all, I would like to thank my supervisor Fredrik. It has been great fun working in your group and I really admire your endless energy and eye for details when it comes to science. I would also like to acknowledge my co-supervisors Marc and Staffan. You have always been very enthusiastic about my work and have provided valuable input when needed. Also, a special thanks to Johan. I don’t think this would have been possible without your help during the first years of my studies. Thanks for always letting me bug you with questions, it has meant a lot to me.

Dicty group: Several people have come and gone during the years and I have enjoyed working with all of you. But I would especially like to thank Maria, Zhen and Bart. Maria, thanks for getting me started in lab, we made a good team! Zhen and Bart, I have really enjoyed sharing office with you. Thank you for all the help and collaborations as well as the many great talks during lunches and coffee breaks. I wish you both the best of luck in the future!

The MOG II crew: Andrea and Benjamin, I really enjoyed developing the course and teaching with you. Also, Dana and Cedric, I’m glad I got to teach with you during the last years. Micro: To all past and present colleagues, there are so many of you that I would like to acknowledge that I don’t know where to start. Micro has been a great place to work and it’s mainly because of you who work there. Regardless if I have needed help with science or just someone to have lunch with, there has always been someone there. Thank you all! Slutligen så vill jag tacka min familj. Mamma, pappa, syster och bror. Jag vet att ni alltid finns där och det har varit ett stort stöd! Bonusfamilj, inklusive svärföräldrar, - Tack för ert intresse och all hjälp genom åren! Allra mest så vill jag tacka Johanna och Fred. Jag är så otroligt glad för att jag har er. Tack för att ni har delat hela den här resan med mig. Ni är bäst!

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