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

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

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

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

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

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