Schematic representation of Linker 2 and the analysed mutations.

<p>Linker 2 is positioned between RBD2 and RBD3. Numbers refer to amino acid residue positions in the <i>S</i>. <i>cerevisiae</i> Mrd1p. Linker definition and consensus sequence as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175506#pone.0175506.ref010" target="_blank">10</a>]. The most conserved part of Linker 2, between amino acid residues 439 and 524, contains four predicted α-helical regions (green boxes) and one β-strand (purple box). The <i>S</i>. <i>cerevisiae</i> Linker 2 amino acid sequence shared by homologues in other species is shown in bold. The conserved WN and AVK/R clusters are underlined. Single amino acid residue substitutions (W458A, K480E and K494E) are indicated together with three extensive mutations (Swap 1, Swap 2, Scrambled). In WNsubst and AVKsubst, the substitutions are shown in red and their locations are pointed out above the schematic representation of secondary structure predictions. In two additional mutants, 85 amino acid residues (curved line) were inserted, either between RBD2 and Linker 2 (5′-ins) or between Linker 2 and RBD3 (3′-ins).</p

Growth characteristics of Linker 2 mutants and expression of Mrd1 mutant proteins.

<p>A. Mutant <i>MRD1</i> genes were introduced into yeast cells, either as part of a plasmid (P_GAL<i>MRD1</i>+plasmid) or integrated into the genome (P_GAL<i>MRD1</i>+Genomic). In both cases, a WT <i>MRD1</i> gene was present in the genome, controlled by a <i>GAL1</i> promoter. The cells were diluted in steps (10 times for each step) and pipetted onto agar plates (from left to right), containing either galactose or glucose, followed by incubation at 30°C, 37°C or 16°C. Mutants are indicated to the left of each dilution series. The parental strain <i>mrd1-ΔL2</i>::<i>klURA3</i>, (FLY002, See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175506#pone.0175506.s003" target="_blank">S1 Table</a>), bearing a non-functional <i>MRD1</i> gene, served as a negative control for cell growth. B. Western blot analyses of the expression of Mrd1p in WT and mutant strains. Extracts from approximately 5x10⁵ cells were used. Ponceau staining demonstrated that approximately equal amounts of total proteins was loaded in each well (data not shown). The WT or mutant <i>MRD1</i> genes were either present in the genome or in a plasmid. The protein A part of the HTP tagged proteins was used for detection. WT Mrd1p is 101 kDa and its HTP tag is approximately 17 kDa. Migration of size marker proteins are shown (180, 130, 100, 70, kDa).</p

Sucrose gradient centrifugation analyses of Linker 2 mutants.

<p>Extracts from cells expressing WT Mrd1p or mutated Mrd1 proteins were centrifuged in a 10–50% a sucrose gradient and fractions were collected. Absorbance at 260 nm and positions of 25S and 18S rRNAs determined by Northern analyses were used to locate 80S ribosomes and 60S and 40S ribosomal subunits. Proteins in each fraction were analysed by Western blot. The tagged Mrd1 proteins were detected with anti-protein A antibody. Migration of size marker proteins is shown to the left (180, 130, 100, 70, kDa).</p

Sequence conservation characteristics of the individual RBDs.

<p>A. Consensus sequences of RBDs 1–6. The conserved residues are ordered according to frequency, with the most frequently occurring amino acid residue at the top. Rbm19 (human) residues are shown in bold. Secondary structure elements are derived as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043786#pone-0043786-g001" target="_blank">Fig. 1B</a>. B. Extent of conservation along each RBD. A window of five residues was slided along each of the six consensus RBDs, calculating the average presence of conserved residues (0–1, y-axis). This value is assigned to the central position of the window (solid line). The positions of α-helices are indicated in green and β-strands in red. C. Conserved residues in the 3D-structures of RBD2–6. Ribbon diagrams showing the 3D-structures of the human RBD2 (PDB identifier 2DGW) and the mouse RBD3–6 (PDB identifiers 1WHW, 1WHX, 2CPF and 2CPH, respectively), all in the same orientation, facing the β-sheet and with loops 1, 3 and 5 pointing downwards. Conserved residues are shown in red. In each RBD, the α1- and α2-helices as well as the β-strands (β1–β4) are labelled.</p

Properties of the linker regions.

<p>A. Disorder prediction of Rbm19 (human) and Mrd1 (<i>S. cerevisiae</i>). Grey areas indicate the RBDs: the red line represents the 0.05 threshold above which values are considered to indicate disorder. RBDs (RBD1–6) and linkers (L1–5) are indicated. B. Consensus sequence of linker 3. Conserved residues are ordered according to frequency, with the most common residue at the top. Rbm19 (human) residues are in bold. The secondary structure prediction given above the sequence (H = α-helix) is for linker 3 in Rbm19 (human).</p

Mutational analysis of loop 5-β4 in RBD6 of Mrd1.

<p>A. The amino acid sequence of loop 5-β4 is shown for RBD6 and RBD5 of Mrd1 in comparison with the corresponding consensus sequences and secondary structure predictions. The analysed amino acid residues are shown in red. B. Growth characteristics of mutant <i>S. cerevisiae</i> cells compared to wild type. A dilution series (from left to right) of each mutant cell was spotted onto a selective FAA agar plate. The relevant amino acid sequence for each strain is shown to the right. Residues in red indicate the tested amino acid substitutions. The last mutant strain has the RBD6 sequence exchanged for the corresponding RBD5 sequence. C. Northern blot analysis of rRNA and pre-rRNA in the two mutant cells with impaired growth. The wild type <i>MRD1</i> gene under the control of a GAL promoter was shut off by growth in glucose medium for the indicated number of hours. Top panel shows the levels of 25S and 18S rRNA are shown after methylene blue staining of the membrane. Bottom panel shows the levels of the 35S, 23S and 20S pre-rRNAs are shown after hybridization with an oligonucleotide probe.</p

Phylogenetic tree of eukaryotic lineages showing Rbm19/Mrd1 proteins containing five or six RBDs.

<p>The tree is redrawn from <a href="http://tolweb.og/Eukaryotes/" target="_blank">http://tolweb.og/Eukaryotes/</a>. Blue lines indicate organisms with five RBDs and green lines those with six RBDs. Grey lines indicate branches for which no completely sequenced genome is known yet. Dashed lines indicate uncertainties in the tree topology. Microsporidia are fungi, but exceptionally have only four RBDs.</p

Dendrogram showing relationships between the RBDs.

<p>The six different RBDs form six clearly separated clusters, of which RBDs 1, 5 and 6 are most easily discernible. Each RBD is denoted with a two-letter code for species and a digit for the RBD number. Sequences are taken from: hs – <i>Homo sapiens</i> (Q9Y4C8), da – <i>Drosophila ananassae</i> (B3MYP1), ss – <i>Salpingoeca sp</i> (F2U536), mb – <i>Monosiga brevicollis</i> (A9USE7), cb – <i>Caenorhabditis briggsae</i> (A8WV73), bd – <i>Batrachochytrium dendrobatidis</i> (F4NSW1), dd – <i>Dictyostelium discoideum</i> (Q54PB2), tp – <i>Thalassiosira pseudonana</i> (B8BZC4), pi – <i>Phytophthora infestans</i> (D0NJ71), es – <i>Ectocarpus siliculosus</i> (D8LH81), at – <i>Arabidopsis thaliana</i> (F4JT92), ol – <i>Ostreococcus lucimarinus</i> (A4RVV1), tg – <i>Toxoplasma gondii</i> (B6KPW8), pm – <i>Perkinsus marinus</i> (C5KH14), sc – <i>Saccharomyces cerevisiae</i> (Q06106), pe – <i>Paramecium tetraurelia</i> (A0DWV5), ed – <i>Entamoeba dispar</i> (B0ECZ6), tc – <i>Trypanosoma cruzi</i> (E7KXH4), ng – <i>Naegleria gruberi</i> (D2V9G7), co – <i>Capsaspora owczarzaki</i> (E9C5E6), gl – <i>Giardia intestinalis</i> (A8BKE6). The number after each abbreviation indicates the RBD position.</p

Alignment of the microsporidia Rbm19/Mrd1 homologues to <i>S. cerevisiae</i> Mrd1.

<p>Alignment of RBDs 1, 3, 4, 6 and linker 3 of <i>S. cerevisiae</i> Mrd1 (denoted y) to <i>E. bieneusi</i> Mrd1 (denoted e). Identical residues (dark grey) or similar (light grey) between the two homologues are indicated. Secondary structure predictions are shown above the sequences. Positions present in the general consensus sequences (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043786#pone-0043786-g005" target="_blank">Fig. 5A</a>) are underlined and asterisks indicate where 2 out of 3 of the microsporidia homologues (B7XJ60, C4V7E1 and E0S816) are conserved. Q8SRD9 was excluded due to high sequence similarity to E0S816 (>80% in the RBDs), in order to avoid bias.</p