Subcellular localization of RbdB GFP and co-localization with DrnB.

<p>AX2 cells were transformed with the integrating plasmid pDneo2a RbdB GFP and subcellular localization was analyzed by fluorescence microscopy. A: Living cells were analyzed in low fluorescence axenic medium showing a diffuse distribution of the fusion proteins in the nucleoplasm and distinct foci at the periphery of the nuclei. Scale bar represents 5 μm. B: To better localize the subnuclear foci, cells were fixed with methanol and analyzed by an OptiGrid microscope (Leica DM 5500). Genomic DNA was stained by DAPI (red). The nucleoli showed no or only a very weak staining. Merging GFP (green) and DAPI (red) signals indicated that RbdB-GFP foci were enriched adjacent to areas with weak or no DAPI staining. Scale bar represents 2.5 μm. C: Co-localization of GFP DrnB and RbdB mRFP in nucleoli associated foci was monitored by fluorescence microscopy using methanol fixed cells. Shown is a single nucleus. Fusion proteins were expressed from extrachromosomally replicating plasmids. Scale bar represents 2.5 μm.</p

Schematic representation of dsRBD containing proteins in <i>D</i>. <i>discoideum</i>.

<p><b>Dhx9</b> (1472 aa: dsRBD (1) [365–440], dsRBD (2) [532–607], DEXDc [715–903], HELICc [963–1069], HA2 [1132–1243]), <b>HelF</b> (837 aa: dsRBD [2–76], DEXDc [228–431], HELICc [608–687]), <b>RbdA</b> (297 aa: dsRBD [4–70]), <b>RbdB</b> (733 aa: dsRBD [9–75]), P-rich [510–584]. Numbers in brackets indicate the position of protein domains in the amino acid (aa) sequence predicted by SMART [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006057#pgen.1006057.ref045" target="_blank">45</a>]. DsRBD (<i>double stranded</i> RNA <i>binding domain</i>), DEXDc (<i>Dead-like Helicases superfamily domain</i>), HA2 (<i>Helicase associated domain</i> 2), P-rich site (Proline rich site). Domains are drawn to scale.</p

Co-IP of RbdB Δ 504–612 GFP and DrnB 3xHA.

<p>RbdB (Δ 504–612) GFP and DrnB 3xHA were expressed in the AX2 wt background. Co-immunoprecipitation of DrnB 3xHA by GFP tagged RbdB (Δ 504–612) was performed. Different samples (IN = input, Pre = preclear, SN = supernatant, E = elution) were analyzed by Western Blots. The fusion proteins were detected by specific α-GFP and α-3xHA antibodies. Numbers indicate the percent of input that was loaded on the SDS-gel. Control IPs were performed with strains expressing the nuclear localized HcpA GFP + DrnB 3xHA or RbdB (Δ504–612) GFP + HcpA 3xHA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006057#pgen.1006057.s003" target="_blank">S3 Fig</a>).</p

Analysis of truncated RbdB variants.

<p>A: Schematic representation of RbdB and truncated protein variants. In RbdB Δ504–733 GFP, 230 amino acids were deleted from the C-terminus. RbdB Δ504–612 GFP lacks the Prich-site. B: left: Both truncated RbdB-GFP versions were expressed in the knockout background and visualized by fluorescence microscopy. RbdB Δ504–612 GFP showed the same distribution as RbdB GFP. In contrast, RbdB Δ504–733 GFP was not detectably by fluorescence microscopy. right: Western Blot showing expression of RbdB Δ504–733 GFP (84 kDa), RbdB Δ504–612 (98 kDa) and of RbdB GFP (109 kDa). Note that all proteins run at higher molecular levels than calculated. SevA (40 kDa) is shown as loading control. C: left: Northern Blot analysis of rbdB- strains expressing RbdB GFP [R1], RbdB Δ504–733 GFP [R2], RbdB Δ504–612 GFP [R3] on miRNAs. 12 μg total RNA were loaded per lane. As a control, RNA from the AX2 wt and from an rbdB- strain was used. The mature miRNAs ddi-miR-1176 and ddi-miR-1177 were detected by ³²P labelled probes as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006057#pgen.1006057.g003" target="_blank">Fig 3A</a>. Hybridisation to snoRNA DdR6 was used as a loading control. Right: miRNA signals (ddi-miR-1176 left, ddi-miR-1177 right) were quantified relative to DdR6 from different Northern Blots and normalized to the AX2 wt. R1-R3: rbdB- mutants were transformed with pDM323 RbdB Δ504–733 GFP (R1), pDM323 RbdB Δ504–612 GFP (R2) and with pDM323 RbdB GFP (R3). According to paired t-test, no significant difference was seen in miRNA accumulation between the wild type and the mutants.</p

Subcellular localization of the Serrate ortholog (SrtA) in <i>D</i>. <i>discoideum</i>.

<p>A: Protein structure of the <i>D</i>. <i>discoideum</i> Serrate ortholog SrtA. RRM: RNA recognition motif domain, Arsenite-R_2: Arsenite-resistance protein 2, C2H2: Zinc finger domain [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006057#pgen.1006057.ref043" target="_blank">43</a>]. B: AX2 cells expressing Srt GFP fusion proteins were fixed with methanol and analyzed by immunofluorescence. DNA was stained by DAPI (red). GFP (green) and DAPI signals were merged. C: ddi-miR-1176 miRNA processing was analyzed in AX2 and in srtA [RNAi 1] and srtA [RNAi 2] knockdown strains. 12 μg total RNA were loaded per lane. Mature ddi-miR-1176 was detected as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006057#pgen.1006057.g003" target="_blank">Fig 3A</a>. To show equal loading, the membrane was rehybridized with a probe directed against the snoRNA DdR6. D: The expression level of ddi-miR-1176 was quantified relative to DdR6 and normalized to the AX2 wt. Error bars: mean with SD, paired t-test: ddi-miR-1176: AX2/srtA [RNAi] p < 0, 0001 (***).</p

Mature miRNAs in rbdA and rbdB- strains.

<p>Expression levels of ddi-miR-1176 (A, left) and ddi-miR-1177 (B, left) were determined by Northern Blot analysis in the indicated knockout mutants. 12 μg total RNA were loaded per lane. Mature miRNAs were detected by ³²P labeled oligonucleotides #2601 (α ddi-miR-1176) and #2602 (α ddi-miR-1177). To show equal loading, the membranes were rehybridized with a ³²P labeled probe (#2654) against the snoRNA DdR6. The expression levels of ddi-miR-1176 (A, right) and ddi-miR-1177 (B, right) were quantified based on independent Northern Blots. Quantification of miRNA expression is given relative to DdR6 and was normalized to the AX2 wt (= 1). Error bars: mean with SD, paired t-test: ddi-miR-1176: AX2/rbdB- p < 0,0001 (***), AX2/drnB- p < 0,0001 (***), AX2/agnA- p < 0,0001 (***). Ddi-miR-1177: AX2/rbdB- p < 0,0001 (***), AX2/drnB- p < 0,0001 (***), AX2/agnA- p = 0,0026 (**). Number of n is given in the graph. For each mutant strain, at least two biological replicates were analyzed.</p

Domain architecture of RNase III enzymes.

<p>Schematic domain architecture of human Drosha and Dicer, DCL1 from <i>A</i>. <i>thaliana</i> (A.th.) and DrnA and DrnB from <i>D</i>. <i>discoideum</i> (D.d.) is shown. DsRBD (double stranded RNA binding domain), RIII (Ribonuclease III domain), HELICc (helicase superfamily c-terminal domain), DEXDc (Dead-like Helicases superfamily domain), PAZ (PAZ-domain), DUF283 (DUF283 domain). Human Drosha contains a region that is rich in prolines (P-rich site) as well as a region that is rich in serine and arginine (S/R-rich). DrnA and DrnB lack the Helicase domains, the DUF283 domain as well as the PAZ domain. DrnB exhibits a proline and serine rich (P/S-rich) region. Proteins and Domains are drawn to scale.</p

dsRBD containing proteins in <i>D</i>. <i>discoideum</i> (AX4).

<p><i>D</i>. <i>discoideum</i> proteins with dsRBDs were identified by an InterPro search [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006057#pgen.1006057.ref043" target="_blank">43</a>]. Ribosomal and translation related proteins are shown in grey.</p

RbdB GFP fusion proteins fully complement the mutant phenotype.

<p>Independent rbdB- strains that were transformed with the plasmid pDbsr2a RbdB GFP [LC, low copy] or pDneo2a RbdB GFP [HC, high copy] were analyzed with respect to miRNA expression A: miRNA expression were analyzed by Northern Blot. 12 μg total RNA were loaded per lane. One clone with low RbdB GFP expression and one clone with high expression each are shown. As controls, we loaded RNA from the AX2 wt and from rbdB- strains. Mature miRNAs were detected by specific ³²P labelled probes as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006057#pgen.1006057.g003" target="_blank">Fig 3A</a>. As a loading control, the membrane was finally rehybridized with a probe directed against the snoRNA Ddr6. B: miRNA signals from independent Northern Blots were quantified relative to the loading control and normalized to the AX2 wild type. According to paired t-test, no significant difference in expression levels was observed between the wild type and the different rescue strains.</p

primary miRNAs accumulated in rbdB- and drnB- strains.

<p>A: Schematic representation of ddi-miR-1176 and ddi-miR-1177 transcripts and predicted pre-miRNA structures. Arrows indicate primers that were used to amplify primary miRNA transcripts. As a control, RT-PCR on <i>corA</i> mRNA was performed. B: Gene specific (reverse) primers were used to generate cDNA molecules: #1828 (<i>corA</i>), DM059 (pri-ddi-miR-1176), DM083 (pri-ddi-miR-1177). The primer sets P1/P2 (DM058/DM059), P2/P3 (DM082, DM083) and #1828/#1829 were used in the following PCR reaction to amplify pri-ddi-miR-1176 (390 bp), pri-ddi-miR-1177 (283 bp) and <i>corA</i> (200 bp) fragments respectively. The number in brackets indicates number of PCR-cycles. For unknown reasons, the minus RT control for pri-ddi-miR-1176 in the AX2 wild type always produced a weak signal.</p