Reconstruction of broadly distributed RNA repertoires for each domain, plus interdomain RNA families.

<p>Colored bars at far right indicate normalized taxonomic abundance of each Rfam for major taxonomic groupings within each domain. Horizontal traces (see text, Table S1 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002752#pcbi.1002752.s008" target="_blank">Text S1</a>) for interdomain families, are depicted as follows: general transfer patterns are given by dashed arrows; proposed HGT patterns for individual families are depicted by number (inset). For Rfam families present in more than one domain (far left and inset), bars indicate normalized taxonomic abundance by domain (color scheme at bottom left). Asterisks indicate additional broadly-distributed bacterial candidates identified using GEBA tree topology <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002752#pcbi.1002752-Wu1" target="_blank">[56]</a> (see text). Note that the Rfam rRNA families in Rfam 10.0 are based on conserved subsequences, and are not as comprehensive as other resources (see main text) and are included here for consistency. The universally-distributed rRNAs are the small subunit (16/18S) rRNA, large subunit (23/28S) rRNA and 5S rRNA (see Table S1 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002752#pcbi.1002752.s008" target="_blank">Text S1</a>). The 5.8S rRNA of eukaryotes is known to be homologous to the 5′ end of bacterial and archaeal 23S rRNA <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002752#pcbi.1002752-Jacq1" target="_blank">[74]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002752#pcbi.1002752-Lafontaine1" target="_blank">[75]</a>, so its inclusion as a eukaryote-specific family in Rfam is in this respect artefactual.</p

Rfam-based functional classification of RNA families.

<p>The tree depicts classification of the higher level data structures within Rfam, and is not a phylogeny. Numbers of sequences and families in Rfam 10 that fall into each functional classification are shown as bar charts. Domain-level taxonomic distribution for each functional category is shown by black (present) and white (absent) boxes, right. The grey box indicates that H/ACA family RNAs are known from archaea <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002752#pcbi.1002752-Tang1" target="_blank">[47]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002752#pcbi.1002752-Muller1" target="_blank">[48]</a>, but are not in Rfam 10.</p

RNA-based processes traceable to the Last Universal Common Ancestor.

<p>Universal Rfam families that show evidence of vertical inheritance (Table S1 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002752#pcbi.1002752.s008" target="_blank">Text S1</a>) are all associated with the processes of translation (rRNAs, tRNAs, RNase P) and protein export (SRP RNA). A previous study examining the antiquity of protein coding genes <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002752#pcbi.1002752-Harris1" target="_blank">[21]</a> identified only 37 universally distributed proteins which show evidence of vertical inheritance. The majority of these vertically inherited proteins are associated with translation and protein export; numbers of such proteins associated with each of the depicted processes is given in grey (original data are from Harris <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002752#pcbi.1002752-Harris1" target="_blank">[21]</a>). The proteins associated with RNase P are not universally conserved, with archaeal and eukaryotic RNase P proteins being unrelated to their bacterial counterparts <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002752#pcbi.1002752-Hartmann1" target="_blank">[72]</a>. While tRNA synthetases are universal, they have undergone ancient horizontal gene transfer events <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002752#pcbi.1002752-Fournier1" target="_blank">[73]</a>, which complicates establishing the timing of their origin.</p

Venn diagram of RNA family distribution.

<p>Taxonomic information attached to EMBL-derived Rfam annotations reveals that the majority (99%) of RNA families are domain-specific, with only seven RNA families universally conserved (across the three domains of life plus viruses; Table S1 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002752#pcbi.1002752.s008" target="_blank">Text S1</a>). Numbers within dashed circles indicate viral RNA families.</p

Tombusvirus genome structure and p19 crystal structure.

(A) Schematic diagram showing the arrangement of the tombusvirus genome, including the overprinting of p19 on the movement protein (MP). (B) Crystal structure of the tomato bushy stunt (TBS) virus p19 dimer (PDB ID 1R9F) [24] bound to a 19 bp RNA fragment. The protein subunits are drawn in cartoon style and coloured dark and light grey; the two RNA strands are drawn as van der Waals spheres and coloured red and orange. Residues found to be under positive selection in at least two analyses are coloured cyan (cartoon representation), and residues identified as being under positive selection by all four analyses are drawn explicitly and coloured according to atom type (cyan: carbon; red: oxygen; blue: nitrogen; white: hydrogen). The dashed square indicates the region shown in panel C. (C) Close up view of the dimer interface with residues Arg139 and Glu143 of each subunit drawn explicitly.</p

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

Potential energies and number of hydrogen bonds between different components of simulated p19 systems.

<p>Systems comprise wild-type and all <i>permissible</i> mutations of p19 alone or in complex with the siRNA. (A) Average potential energies (<i>E</i>_pot) of the interaction between the two protein subunits making up the p19 dimer (prot:prot). (B) Average potential energies (<i>E</i>_pot) of the interaction between the protein dimer and the RNA (prot;RNA). (C) Average potential energies (<i>E</i>_pot) of the complete protein/RNA complex (prot+RNA). The electrostatic and van der Waals contributions are shown separately alongside the total potential energy, as indicated above the graphs. (D) Average number of hydrogen bonds during the entire simulation between the two protein subunits making up the p19 dimer. (E) Average number of hydrogen bonds during the entire simulation between the protein dimer and the RNA. Solid bars correspond to simulations with RNA included, and empty bars to simulations without RNA present. Averages were calculated from the first 50 ns of simulation, after 10 ns equilibration, so that the averages calculated from the simulations of p19 with RNA bound (200 ns) are comparable to those of the simulations without RNA bound (50 ns). The error bars correspond to the standard deviation in all cases.</p

Residues identified as being under positive selection.

<p>See text for details of each of the four analyses. Light green indicates residues that only some of the analyses identified as being under positive selection. Dark green indicates residues found to be under positive selection in all four analyses. Note that some residues predicted to be under positive selection by HyPhy FEL2 (36, 43, 67, 107, 145) actually show no variation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147619#pone.0147619.g002" target="_blank">Fig 2</a>).</p

Alignment of amino acid sequences of p19 from 11 different tombusvirus species.

<p>The single-letter residue codes are coloured according to the nature of the amino acid side chain: (red) negatively-charged (D,E); (dark blue) aromatic (F,Y); (blue) positively-charged (K, R); (cyan) large polar amide-containing (Q,N); (orange) small polar hydroxyl-containing (S,T); (yellow) sulfur-containing (C,M); (grey) small aliphatic (A,G); (green) medium aliphatic (I,V,L); (purple-blue) imidazole (H); (violet) indole (W); (pink-brown) cyclised secondary amine (P). Sites identified as being under positive selection by all four analyses are highlighted in pale green. The secondary structure elements are indicated above the sequences: (barrels) α-helices; (arrows) β-strands. Viral species and NCBI accession numbers are as follows: CIR (Carnation italian ringspot virus, NC003500), TBS (Tomato bushy stunt virus, NC001554), AMC (Artichoke mottle crinkle virus, NC001339), LNV (Lisianthus necrosis virus, NC007983), PLV (Pear latent virus, NC004723), PNV (Pelagornium necrotic streak virus, NC005285), CNV (Cucumber necrosis virus, NC001469), GAL (Grapevine algerian latent virus, AY830918), CRV (Cymbidium ringspot virus, NC003532), CBL (Cucumber bulgarian latent virus, NC004725), MNV (Maize necrotic streak virus, NC007729).</p

Summarizing image showing the transformation from the motile trophozoite via encyzoite to the final cyst stage.

The trophozoite monitors the external environment and encystation is induced via intracellular pathways that remain largely unknown. The cell passes a “point of no return” during early encystation after which it is no longer possible to relapse to the proliferating stage. Transcription factors (e.g. Myb2) activates encystation-specific genes among them are cyst wall proteins (CWP1-3). An overall increase of translation could be observed early in encystation as the production of CWPs is dramatically increased and the transportation in encystation vesicles (ESVs) begins. The vesicles undergo maturation steps after leaving the ER. The other component of the cyst wall, the UDP-GalNAc sugar (giardin), is also synthesized and secreted via encystation positive carbohydrate vesicles (ECVs). The enzymes involved in giardin synthesis are induced during encystation. During late encystation, the cell changes shape as it enters dormancy and the ventral disc together with the flagella are disassembled as the construction of the cyst wall proceeds. But the mechanism behind the assembly is still unknown. Often pre-cyst stages with a “tail” can be observed in encystation. Two rounds of DNA replication occur without cytokinesis rendering a cyst with four nuclei each with the genome ploidy of 4N. Interconnections between the nuclei in the cysts are formed and genetic material can be exchanged through the process “diplomixis”. During excystation, each cell receives one pair of non-sister nuclei (indicated as red and blue).</p