Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba <i>Dictyostelium discoideum</i>

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 3ʹ-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.</p

MOESM3 of Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model

Additional file 3. Differentially regulated genes in D. discoideum one and six hours post L. pneumophila infection. Regulation and description of all D. discoideum genes regulated (FDR < 0.05) one and six hours post L. pneumophila infection. The regulation previously determined by microarray for these genes are also included

MOESM2 of Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model

Additional file 2. Differentially regulated genes in D. discoideum 2.5 h post M. marinum infection. Regulation and description of all D. discoideum genes regulated (FDR < 0.05) 2.5 h post M. marinum infection

MOESM1 of Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model

Additional file 1. Supplementary figures, tables and additional results. Figure S1. Cytotoxic effect and fraction of infected cells at different multiplicity of infection (MOI) with M. marinum. Figure S2. Validation of RNA-seq by RT-qPCR when challenged with M. marinum. Figure S3. Regulation detected with microarray vs corresponding RNA-seq values. Figure S4. Comparison of transcriptional response to L. pneumophila infection one and six hours post infection. Figure S5. Regulation of overlapping genes in D. discoideum in response to M. marinum, L. pneumophila and E. coli. Table S1. Genes discussed in Additional results and in the connected Result section. Table S2. Primer sequences and annealing temperatures used for the RT-qPCR analyses. Additional results – Additional description of D. discoideum response to M. marinum and L. pneumophila including key genes and functions

MOESM4 of Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model

Additional file 4. Enriched GO terms for genes differentially expressed 2.5 h post M. marinum infection. All enriched GO terms including statistics and associated genes

MOESM7 of Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model

Additional file 7. Identified human orthologues for D. discoideum genes regulated in response to infection by mycobacteria, L. pneumophila or both. Gene IDs, regulation and KEGG pathway analyses for orthologous genes

MOESM5 of Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model

Additional file 5. Enriched GO terms for genes differentially expressed one and six hours post L. pneumophila infection. All enriched GO terms including statistics and associated genes

MOESM6 of Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model

Additional file 6. Comparison of D. discoideum genes regulated in response to pathogenic bacteria and food bacteria. Regulation and description of genes included in Fig. 6