Evolution of microRNAs in Amoebozoa and implications for the origin of multicellularity Repository accompanying "Evolution of microRNAs in Amoebozoa and implications for the origin of multicellularity" manuscript microRNA curation Potential miRNA clusters, generated using Shortstack (https://github.com/MikeAxtell/ShortStack), are analyzed with miRNA_curation.py. An example run of the miRNA curation can be performed by running the 'example_run/explore_all.sh' and 'example_run/refine_all.sh' scripts in order. A sample of the sRNA sequences from D. discoideum is included in the 'example/example_sizeselected_data' folder. A sample of miRNA candidates are included in 'example_run/pot-miRNAs_merged.fa' to facilitate the example run. Prerequisites: Python 3, Biopython, RNAlib, matplotlib, numpy microRNA analysis Following miRNA curation, high confidence miRNAs are identified and further analysed in amoeba_miRNAevo.Rmd. The data for the analysis is located in 'data_in' and are mostly derived from the miRNA_curation.py script and other scripts located in the 'helper_scripts' folder. The plots generated in the analysis is located in the 'plots' folder. Tables and other data generated from the analysis are located in the 'data_out' folder. In order to perform the miRNA conservation search, refer to the 'helper_scripts/miRNA_conservation_search/' folder. Access genomes/annotations For Dictyostelium firmibasis, the genome was de-novo sequenced and is located in 'data_in/novel_assembly'. Assembly was done using Flye v2.9.1 in nano-hq mode, and two rounds of long-read polishing were done using Medaka v1.7.2. The filtered reads used for assembly can be accessed from NCBI BioProject PRJNA972620. The Acanthamoeba castellanii genome and annotation were accessed at https://www.doi.org/10.5281/zenodo.680005. The other genomes and annotations for this study were accessed from NCBI and can be downloaded using the 'get_references.sh' script located in the helper_scripts folder.

ABSTRACTGastrointestinal disease caused by Salmonella enterica is associated with the pathogen’s ability to replicate within epithelial cells and macrophages. Upon host cell entry, the bacteria express a type-three secretion system encoded within Salmonella pathogenicity island 2, through which host-manipulating effector proteins are secreted to establish a stable intracellular niche. Transcription of this intracellular virulence program is activated by the PhoPQ two-component system that senses the low pH and the reduced magnesium concentration of host cell vacuoles. In addition to transcriptional control, Salmonella commonly employ RNA-binding proteins (RBPs) and small regulatory RNAs (sRNAs) to regulate gene expression at the post-transcriptional level. ProQ is a globally acting RBP in Salmonella that promotes expression of the intracellular virulence program, but its RNA repertoire has previously been characterized only under standard laboratory growth conditions. Here, we provide a high-resolution ProQ interactome during conditions mimicking the environment of the Salmonella-containing vacuole (SCV), revealing hundreds of previously unknown ProQ binding sites in sRNAs and mRNA 3′UTRs. ProQ positively affected both the levels and the stability of many sRNA ligands, some of which were previously shown to associate with the well-studied and infection-relevant RBP Hfq. We further show that ProQ activates the expression of PhoP at the post-transcriptional level, which, in turn, leads to upregulation of the intracellular virulence program.IMPORTANCESalmonella enterica is a major pathogen responsible for foodborne gastroenteritis, and a leading model organism for genetic and molecular studies of bacterial virulence mechanisms. One key trait of this pathogen is the ability to survive within infected host cells. During infection, the bacteria employ a type three secretion system that deliver effector proteins to target and manipulate host cell processes. The transcriptional regulation of this virulence program is well understood. By contrast, the factors and mechanisms operating at the post-transcriptional level to control virulence gene expression are less clear. In this study, we have charted the global RNA ligand repertoire of the RNA-binding protein ProQ during in vitro conditions mimicking the host cell environment. This identified hundreds of binding sites and revealed ProQ-dependent stabilization of intracellular-specific small RNAs. Importantly, we show that ProQ post-transcriptionally activates the expression of PhoP, a master transcriptional activator of intracellular virulence in Salmonella.

All organisms have to carefully regulate their gene expression, not least during development. mRNA levels are often used as proxy for protein output; however, this approach ignores post-transcriptional effects. In particular, mRNA-protein correlation remains elusive for organisms that exhibit aggregative rather than clonal multicellularity. We addressed this issue by generating a paired transcriptomics and proteomics time series during the transition from uni-to multicellular stage in the social ameba Dictyostelium discoideum. Our data reveals that mRNA and protein levels correlate highly during unicellular growth, but decrease when multicellular development is initiated. This accentuates that transcripts alone cannot accurately predict protein levels. The dataset provides a useful resource to study gene expression during aggregative multicellular development. Additionally, our study provides an example of how to analyze and visualize mRNA and protein levels, which should be broadly applicable to other organisms and conditions.

Dicytostelium firmibasis is a member of Dictyostelia, a group of social amoebae that upon starvation display aggregative multicellularity where the amoebae transition from uni-to multicellular life. The D. firmibasis genome assembly that is currently available is of limited use due to its low contiguity, large number of undetermined bases, and lack of annotations. Here we used Nanopore long read sequencing, complemented with Illumina sequencing, and developmental transcriptomics as well as small RNA-sequencing, to present a new, fully annotated, chromosome-level D. firmibasis genome assembly. The new assembly contains no undetermined bases, and consists mainly of six large contigs representing the chromosomes, as well as a complete mitochondrial genome. This new genome assembly will be a valuable tool, allowing comprehensive comparison to Dictyostelium discoideum , the dictyostelid genetically tractable model. Further, the new genome will be important for studies of evolutionary processes governing the transition from unicellular to multicellular organisms and will aid in the sequencing and annotation of other dictyostelids genomes, many of which are currently of poor quality.

A hallmark of ribosomal RNA (rRNA) are 2′- O -methyl groups that are introduced sequence specifically by box C/D small nucleolar RNAs (snoRNAs) in ribonucleoprotein particles. Most data on this chemical modification and its impact on RNA folding and stability are derived from organisms of the Opisthokonta supergroup. Using bioinformatics and RNA-seq data, we identify 30 novel box C/D snoRNAs in Dictyostelium discoideum , many of which are differentially expressed during the multicellular development of the amoeba. By applying RiboMeth-seq, we find 49 positions in the 17S and 26S rRNA 2′-O-methylated. Several of these nucleotides are substoichiometrically modified, with one displaying dynamic modification levels during development. Using homology-based models for the D. discoideum rRNA secondary structures, we localize many modified nucleotides in the vicinity of the ribosomal A, P and E sites. For most modified positions, a guiding box C/D snoRNA could be identified, allowing to determine idiosyncratic features of the snoRNA/rRNA interactions in the amoeba. Our data from D. discoideum represents the first evidence for ribosome heterogeneity in the Amoebozoa supergroup, allowing to suggest that it is a common feature of all eukaryotes.

Aggregative multicellularity has evolved multiple times in diverse groups of eukaryotes, exemplified by the well-studied development of dictyostelid social amoebas, for example, Dictyostelium discoideum However, it is still poorly understood why multicellularity emerged in these amoebas while the majority of other members of Amoebozoa are unicellular. Previously, a novel type of noncoding RNA, Class I RNAs, was identified in D. discoideum and shown to be important for normal multicellular development. Here, we investigated Class I RNA evolution and its connection to multicellular development. We identified a large number of new Class I RNA genes by constructing a covariance model combined with a scoring system based on conserved upstream sequences. Multiple genes were predicted in representatives of each major group of Dictyostelia and expression analysis confirmed that our search approach identifies expressed Class I RNA genes with high accuracy and sensitivity and that the RNAs are developmentally regulated. Further studies showed that Class I RNAs are ubiquitous in Dictyostelia and share highly conserved structure and sequence motifs. In addition, Class I RNA genes appear to be unique to dictyostelid social amoebas because they could not be identified in outgroup genomes, including their closest known relatives. Our results show that Class I RNA is an ancient class of ncRNAs, likely to have been present in the last common ancestor of Dictyostelia dating back at least 600 million years. Based on previous functional analyses and the presented evolutionary investigation, we hypothesize that Class I RNAs were involved in evolution of multicellularity in Dictyostelia.

The global RNA-binding protein ProQ has emerged as a central player in post-transcriptional regulatory networks in bacteria. While the N-terminal domain (NTD) of ProQ harbors the major RNA-binding activity, the role of the ProQ C-terminal domain (CTD) has remained unclear. Here, we have applied saturation mutagenesis coupled to phenotypic sorting and long-read sequencing to chart the regulatory capacity of Salmonella ProQ. Parallel monitoring of thousands of ProQ mutants allowed mapping of critical residues in both the NTD and the CTD, while the linker separating these domains was tolerant to mutations. Single amino acid substitutions in the NTD associated with abolished regulatory capacity strongly align with RNA-binding deficiency. An observed cellular instability of ProQ associated with mutations in the NTD suggests that interaction with RNA protects ProQ from degradation. Mutation of conserved CTD residues led to overstabilization of RNA targets and rendered ProQ inert in regulation, without affecting protein stability in vivo. Furthermore, ProQ lacking the CTD, although binding competent, failed to protect an mRNA target from degradation. Together, our data provide a comprehensive overview of residues important for ProQ-dependent regulation and reveal an essential role for the enigmatic ProQ CTD in gene regulation.

Background: During infection by intracellular pathogens, a highly complex interplay occurs between the infected cell trying to degrade the invader and the pathogen which actively manipulates the host cell to enable survival and proliferation. Many intracellular pathogens pose important threats to human health and major efforts have been undertaken to better understand the host-pathogen interactions that eventually determine the outcome of the infection. Over the last decades, the unicellular eukaryote Dictyostelium discoideum has become an established infection model, serving as a surrogate macrophage that can be infected with a wide range of intracellular pathogens. In this study, we use high-throughput RNA-sequencing to analyze the transcriptional response of D. discoideum when infected with Mycobacterium marinum and Legionella pneumophila. The results were compared to available data from human macrophages. Results: The majority of the transcriptional regulation triggered by the two pathogens was found to be unique for each bacterial challenge. Hallmark transcriptional signatures were identified for each infection, e.g. induction of endosomal sorting complexes required for transport (ESCRT) and autophagy genes in response to M. marinum and inhibition of genes associated with the translation machinery and energy metabolism in response to L. pneumophila. However, a common response to the pathogenic bacteria was also identified, which was not induced by non-pathogenic food bacteria. Finally, comparison with available data sets of regulation in human monocyte derived macrophages shows that the elicited response in D. discoideum is in many aspects similar to what has been observed in human immune cells in response to Mycobacterium tuberculosis and L. pneumophila. Conclusions: Our study presents high-throughput characterization of D. discoideum transcriptional response to intracellular pathogens using RNA-seq. We demonstrate that the transcriptional response is in essence distinct to each pathogen and that in many cases, the corresponding regulation is recapitulated in human macrophages after infection by mycobacteria and L. pneumophila. This indicates that host-pathogen interactions are evolutionary conserved, derived from the early interactions between free-living phagocytic cells and bacteria. Taken together, our results strengthen the use of D. discoideum as a general infection model.

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

RNA interference (RNAi) is a widespread biological process, which regulates gene expression in eukaryotic cells. A complex of proteins and small RNAs, RISC, mediates this gene regulation. Central t ...

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

Continuous communication between cells is necessary for development of any multicellular organism and depends on the recognition of secreted signals. A wide range of molecules including proteins, peptides, amino acids, nucleic acids, steroids and polylketides are used as intercellular signals in plants and animals. They are also used for communication in the social ameba Dictyostelium discoideum when the solitary cells aggregate to form multicellular structures. Many of the signals are recognized by surface receptors that are seven-transmembrane proteins coupled to trimeric G proteins, which pass the signal on to components within the cytoplasm. Dictyostelium cells have to judge when sufficient cell density has been reached to warrant transition from growth to differentiation. They have to recognize when exogenous nutrients become limiting, and then synchronously initiate development. A few hours later they signal each other with pulses of cAMP that regulate gene expression as well as direct chemotactic aggregation. They then have to recognize kinship and only continue developing when they are surrounded by close kin. Thereafter, the cells diverge into two specialized cell types, prespore and prestalk cells, that continue to signal each other in complex ways to form well proportioned fruiting bodies. In this way they can proceed through the stages of a dependent sequence in an orderly manner without cells being left out or directed down the wrong path.

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

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

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

Background: Dictyostelium discoideum encodes both class I and II RNRs. Results: Both RNRs are expressed in D. discoideum and depletion of class I RNR drastically reduces spore formation and viability. Conclusion: Class I RNR is central for D. discoideum growth and differentiation, while class II function is enigmatic. Significance: Our results underscore the evolutionary selection for this rare set of RNRs in D. discoideum.

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

The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs.

Gene silencing may have a direct or indirect impact on many biological processes in eukaryotic cells, and is a useful tool for the determination of the roles of specific genes. In this article, we report silencing in Phytophthora infestans, an oomycete pathogen of potato and tomato. Gene silencing is known to occur in P. infestans, but its genetic basis has yet to be determined. Genes encoding the major components of the RNA interference (RNAi) pathway, Dicer-like (Pidcl1), Argonaute (Piago1-5) and RNA-directed RNA polymerase (Pirdr1), were identified in the P. infestans genome by comparative genomics, together with families of other genes potentially involved in gene silencing, such as histone deacetylases, histone methyltransferases, DEAD heli-cases, chromodomain proteins and a class 1 RNaseIII. Real-time reverse transcription-polymerase chain reaction demonstrated transcript accumulation for all candidate genes throughout the asexual lifecycle and plant infection, but at different levels of mRNA abundance. A functional assay was developed in which silencing of the sporulation-associated Picdc14 gene was released by the treatment of protoplasts with in vitro-synthesized double-stranded RNAs homologous to Pidcl1, Piago1/2 and histone deacetylase Pihda1. These results suggest that the components of gene silencing, namely Dicer-like, Argonaute and histone deacetylase, are functional in P. infestans. Our data demonstrate that this oomycete possesses canonical gene silencing pathways similar to those of other eukaryotes.