Schematic presentation of the knock-down system.

<p>(A) A gene fragment is inserted in between the left and right ITR as a trigger sequence. (B) Genes used for knock-down analysis are presented as mRNA with accession number and sequence length. Trigger sequences are indicated by black bars with position of the first and last nucleotide in respect to the ATG start. Oligonucleotides and their orientation used as hybridization probes are indicated by black arrows.</p

Model for gene silencing by natural and transgene derived siRNAs.

<p>(A) The endogenous DIRS–1 sequences are transcribed in both directions and generate dsRNAs (grey) which are cleaved by Dicer to ~21 nt siRNAs. siRNAs recruit RrpC in a AgnA-dependent manner. The resulting dsRNA which may be synthesised primer dependent or primer independent in 5’ and 3’ direction is processed to secondary siRNAs. Primer independent siRNAs carry a 5’-triphosphate. Argonaute proteins (probably AgnA) silence DIRS–1 by mRNA destabilisation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131271#pone.0131271.ref010" target="_blank">10</a>]. (B) A transgene fusion of GFP and a DIRS-1 ORF is targeted by endogenous DIRS-1 siRNAs <i>in trans</i>. These may be primary or secondary siRNAs. They recruit the RdRP RrpC, which produces primer dependent and primer independent secondary siRNAs in 5’ and 3’ direction. Consequently, siRNAs matching adjacent sequences (here GFP) are generated. Silencing occurs on the level of mRNA destabilisation. DIRS–1 sequences are colour coded grey, GFP sequences green. Secondary siRNAs have a 5’-triphosphate or a monophosphate when they are Dicer products from longer dsRNAs or primer dependent. (C) siRNAs are generated from a bidirectionally transcribed trigger transgene. They can target the trigger transcripts <i>in cis</i> and complementary sequences on mRNAs of the corresponding endogene. They produce secondary siRNAs either from the trigger only or from both the target and the trigger transcript. If transgene derived siRNAs can only recruit RdRPs <i>in cis</i>, i.e. on the transgene itself, this could explain the lack of spreading along the mRNA of the endogene. Trigger sequences are colour coded grey, flanking sequences of the corresponding endogene blue.</p

GFP RNA and protein levels from bidirectional promoter constructs.

<p>(A) Schematic presentation of the GFP reporter constructs. The GFP coding region was inserted between lITR and rITR of DIRS–1 and between opposing actin15 promoters (A15P). As a control, GFP with flanking A15P and A8 terminator (A8T) was used. (B) Analysis of GFP mRNA by Northern blots in the indicated strains. Blots were hybridized with probes against GFP sense mRNA (sense gfp, probe P29) and antisense mRNA (antisense gfp, probe P30). Equal loading was verified by rehybridization of the membrane with a ³²P labelled oligonucleotide directed against actin. Samples for A15P GFP A15P were run on a different gel and hybridized separately (see actin loading control). (C) Representative Northern blot of GFP and DIRS–1 siRNAs in the indicated strains. Equal loading was verified by rehybridization of membrane with a ³²P labelled probe directed against snoRNA DdR-6. (D) Representative Northern blot comparing GFP siRNAs generated from opposing ITRs and opposing A15 promoters. DdR-6 was used as a loading control. (E) Representative Western blot of GFP expression under the control of the left and right ITR in the indicated strains. GFP expression by A15P (¼ was loaded to avoid overexposure of the GFP signal) was used as a positive, the untransformed Ax2 as a negative control. CorA was used as a loading control. Quantification of the GFP intensity was performed using ImageJ. GFP expression by A15P was set to 1 (right panel). Ax2 lITR GFP rITR: n = 2. agnA- lITR GFP rITR: n = 4. Error bars: mean with SD. (F) Comparison of GFP silencing by opposing ITRs and A15Ps. GFP expression by A15P (¼ was loaded to avoid overexposure of the GFP signal) was used as a positive control.</p

Comparison of published knock-out phenotypes with knock-downs by ITR constructs.

<p>(A) Nuclei count in Ax2 wild type cells and different knock-down strains. Knock-down of corA (lTR corA rITR) resulted in increased multinuclearity. 200 cells of each strain of two independent transformations were counted. (B) Knock-down of mhcA by an ITR construct showed huge cells containing up to 30 nuclei and more compared to Ax2 wild type and to a control knock-down of abpA. Scale bar 50μm. (C) Growth curve of Ax2 wild type cells and different knock-down strains. Knock-down of casK resulted in a slow growing culture. Measurement of cell density in the indicated cell lines over 72 hours. Data are plotted as relative cell count over time. Ax2, lITR GFP rITR, lITR abpA rITR: n = 2; lITR casK rITR: n = 4.</p

Detection of siRNAs based on the bidirectional RNA expression driven by the left and right DIRS-1 ITRs.

<p>(A) Northern blot to detect alpha-Actinin (abpA), Coronin (corA) and Severin (sevA) siRNAs. Probes at the trigger-ends and near the trigger-centres are indicated and correspond to the probes shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131271#pone.0131271.g003" target="_blank">Fig 3</a> (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131271#pone.0131271.s003" target="_blank">S1 Table</a>). (B) Northern blot to detect casein kinase (casK), cullin D (culD), queuine tRNA-ribosyl transferase 1 (qtrt1) siRNAs at the end of the trigger. A mixture of radiolabelled oligonucleotides (position indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131271#pone.0131271.g003" target="_blank">Fig 3</a>, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131271#pone.0131271.s003" target="_blank">S1 Table</a>) was used for hybridization. All samples in A and B respectively were run on the same gel. Probes against snoRNA DdR-6 and DIRS–1 siRNAs were separately used as loading controls for A and B.</p

Analysis of ITR promoter activity.

<p>(A) The GFP coding region was inserted between actin15 promoter (A15P) and actin8 terminator (A8T), DIRS–1 left ITR (lITR) and A8T, inverted DIRS-1 right ITR (rITR) and A8T. (B) Detection of GFP mRNA expression in Ax2 wild type driven by the constructs depicted in Fig 1A. As a loading control, the blot was rehybridised with a ³²P labelled oligonucleotide directed against actin (left panel). Western blot shows GFP protein expression in Ax2 wild type driven by the constructs depicted in Fig 1A. Coronin A (CorA) was used as a loading control (middle panel). The right panel shows the corresponding quantification of Western blots. Analysis was performed using ImageJ. Ax2 A15 GFP: n = 8. Ax2 lITR GFP: n = 4. Ax2 rITR GFP: n = 4. Error bars: mean with SD. (C) Detection of mRNA expression (left) and protein level (middle) for GFP driven by the lITR in Ax2 wild type and agnA knock-out (agnA-) cell lines. Numbers in square brackets indicate numbers of independent populations. The right panel shows the corresponding quantification of Western blots. Analysis was performed using ImageJ. Similar results were obtained with the right ITR (data not shown). Ax2 lITR GFP: n = 4. agnA- lITR GFP: n = 4. Error bars: mean with SD.</p

Determination of 5’ endgroups by Terminator 5'-phosphate-dependent exonuclease (5'exo).

<p>RNA samples of the ITR GFP strains (silencing <i>in cis</i>) and ITR abpA strains (silencing <i>in trans</i>) were digested with 5'exo, in buffer A and buffer B as indicated, separated on an acrylamide gel, blotted and probed with radiolabelled oligonucleotides complementary to 5.8S (digestion control), tRNA^Asp (control for undigested RNA) and for GFP and abpA siRNAs respectively.</p

The protein domains of the <i>Dictyostelium</i> microprocessor that are required for correct subcellular localization and for microRNA maturation

<p>The maturation pathways of microRNAs (miRNAs) have been delineated for plants and several animals, belonging to the evolutionary supergroups of Archaeplastida and Opisthokonta, respectively. Recently, we reported the discovery of the microprocessor complex in <i>Dictyostelium discoideum</i> of the Amoebozoa supergroup. The complex is composed of the Dicer DrnB and the dsRBD (double-stranded RNA binding domain) containing protein RbdB. Both proteins localize at nucleoli, where they physically interact, and both are required for miRNA maturation. Here we show that the miRNA phenotype of a Δ<i>drnB</i> gene deletion strain can be rescued by ectopic expression of a series of DrnB GFP fusion proteins, which consistently showed punctate perinucleolar localization in fluorescence microscopy. These punctate foci appear surprisingly stable, as they persist both disintegration of nucleoli and degradation of cellular nucleic acids. We observed that DrnB expression levels influence the number of microprocessor foci and alter RbdB accumulation. An investigation of DrnB variants revealed that its newly identified nuclear localization signal is necessary, but not sufficient for the perinucleolar localization. Biogenesis of miRNAs, which are RNA Pol II transcripts, is correlated with that localization. Besides its bidentate RNase III domains, DrnB contains only a dsRBD, which surprisingly is dispensable for miRNA maturation. This dsRBD can, however, functionally replace the homologous domain in RbdB. Based on the unique setup of the <i>Dictyostelium</i> microprocessor with a subcellular localization similar to plants, but a protein domain composition similar to animals, we propose a model for the evolutionary origin of RNase III proteins acting in miRNA maturation.</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

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