Potential factors involved in snoMEN machinery.

<p>(<b>A</b>) U2OS^{GFP–SMN1-PR} and U2OS^GFP–SMN1 cells were transfected with either Scrambled siRNA, Ago1 siRNA, Ago2 siRNA, Upf1 siRNA, or fibrillarin siRNA. An equivalent amount of each extract was loaded in each lane and the proteins were separated by SDS PAGE, electroblotted onto membrane and probed with anti-SMN1, anti-Ago1, anti-Ago2, anti-Upf1, anti-fibrillarin and with anti-tubulin as a loading control. (<b>B</b>) The graphs show average SMN signal intensity and standard deviation for three independent experiments using the same procedure as in A. SMN signal ratio was normalised to the tubulin signal.</p

Procedure for establishment of human protein replacement stable cell line using snoMEN.

<p>(A) Structures for targeted endogenous SMN1/UBF1 protein replacement plasmids (pGFP–SMN1snoMENv1-PR/pmCherry–UBF1snoMENv1-PR). These constructs have three snoMEN sequences as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062305#pone.0062305-Ono1" target="_blank">[1]</a>, except that the M box sequences are complementary to endogenous SMN1/UBF1 pre-mRNA sequences (See Materials and Methods). (B) Procedure of stable cell line establishment. Transfected cells were selected under G418 treatment as previously described (<a href="http://www.lamondlab.com/f7protocols.htm" target="_blank">http://www.lamondlab.com/f7protocols.htm</a>). Cells were cultured for at least 14 passages before analysis to confirm stable FP–protein and snoMEN expression. (C) Images of protein replacement stable cell lines. Expression of FP proteins was confirmed by fluorescence imaging. Bar length is 10 µm.</p

Characterisation of FP–protein complexes in replacement stable cell lines by Quantitative SILAC Proteomic analysis.

<p>(<b>A</b>) Design of the triple-encoding SILAC pull down experiments (see text). Comparison of mCherry–UBF1 complex, either in the presence, or absence, of low concentration Actinomycin-D (left panel) and comparison of GFP–SMN1 complex either with replacement (U2OS^{GFP–SMN1-PR}), or without replacement (U2OS^GFP–SMN1) (right panel). The SILAC experiments were independently repeated at least four times. (<b>B</b>) SILAC result of mCherry–UBF1 complex pull down assay visualised on a 2D logarithmic graph for all proteins identified. On the x axis, log₂ (M/L ratio) correlates with the enrichment in mCherry–UBF1 IP versus control IP. On the y axis, log₂ (H/L ratio) correlates with the enrichment in mCherry–UBF1 IP with Actinomycin-D treatment versus no-treatment IP. The bait, UBF1, is shown in red, and a blue line separates the proteins whose interaction with UBF1 is increased (above the line)/or decreased (below) after Actinomycin-D treatment. Known interaction partners, which were identified and quantified, are also highlighted. SILAC ratio values of labelled proteins are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062305#pone.0062305.s007" target="_blank">Table S2</a>. (<b>C</b>) Known UBF1 interaction partners, which were identified and quantified. Graph shows fold change of each protein ratio with Actinomycin-D treatment (red) and without treatment (orange) measured from five independent experiments. (<b>D</b>) Comparisons of protein ratios of known interaction partners of SMN1 between over expression stable cell line (U2OS^GFP–SMN1; light green) and protein replacement stable cell line (U2OS^{GFP–SMN1-PR}; green) measured from five independent experiments.</p

Features of snoMEN technology.

<p>Schematic diagram showing differences between the siRNA/shRNA and snoMEN systems. Arrows show promoters for RNA polymerase III (shRNA) and RNA polymerase II (snoMEN), respectively. Red squares show the coding region, e.g. either mCherry cDNA, or endogenous genes. Striped squares show non-coding exon region. The bars show non-coding regions, e.g. introns.</p

SiRNA and shRNA knock-down targeted to endogenous pre-mRNAs.

<p>(A) & (B) The targeted regions on the RNAs for each of the snoMEN vectors used in this study are shown in a schematic diagram. The same pre-mRNA sequence of UBF1 (A)/SMN1 (B) as targeted by the snoMEN vector was targeted by siRNA oligoribonucleotides and shRNA expression plasmids. (C) Western blot analysis for siRNA experiments. Detection of endogenous UBF1 protein levels following transfection of HeLa cells using either Scrambled siRNA (Control: lane1), UBF1 siRNA (siUBF1: lane2), UBF M box siRNA-1 (siUM1: lane3), UBF M box siRNA-2 (siUM2: lane4), and UBF M box siRNA-3 (siUM3: lane5). An equivalent amount of HeLa extract was loaded for each lane and the proteins separated by SDS PAGE, electroblotted onto membrane and probed both with a monoclonal anti-UBF1 antibody and with an anti-tubulin antibody as a loading control. Graph shows UBF1 signal intensity normalised to the tubulin signal measured from three independent experiments. (D) Structure of shRNA expression plasmids. A cDNA producing a short hairpin RNA was subcloned under the U6 RNA polymerase III promoter. Each of the shRNA plasmids encode ZsGreen FP–protein cDNA under CMV promoter regulation as a transfection marker. (E) UBF1 shRNA plasmid and no-endogenous target shRNA plasmid were transfected as a positive and negative control, respectively. UBF1 Mbox shRNA-1 to -3 (shUM1–3) have the same target sequence as UBF1 snoMEN from set1 to set3, respectively (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062305#pone-0062305-g003" target="_blank">Figure 3A</a>). Scale bar, 10 µm. Arrow: cells not showing knock-down, Arrowhead: cells showing knock-down. (F) Western blot analysis for shRNA experiments. Detection of endogenous UBF1 protein levels following transfection of HeLa cells using either Scrambled shRNA (Control: lane1), UBF1 shRNA (shUBF1: lane2), UBF M box shRNA-1 (shUM1: lane3), UBF M box shRNA-2 (shUM2: lane4), and UBF M box shRNA-3 (shUM3: lane5). An equivalent amount of HeLa extract was loaded for each lane and the proteins separated by SDS PAGE, electroblotted onto membrane and probed both with a monoclonal anti-UBF1 antibody and with an anti-tubulin antibody as a loading control. Graph shows UBF1 signal intensity normalised to the tubulin signal measured from three independent experiments.</p

(A) Plot of FRET efficiencies ± SD (mean for 7–14 cells) between ECFP and EYFP fusion proteins measured by FRET acceptor photobleaching

P-values were obtained from the test comparing the FRET efficiencies with and without DRB treatment. (B) FRET between U1 70K and SC35 measured by FLIM. HeLa cells were transfected with EGFP-U1 70K and cotransfected with either mCherry-C1 or mCherry-SC35. Confocal images are of transfected cells and FLIM images are of the same cells, in which FRET efficiency and FRET amplitude are shown in pseudocolor. The color scale with the respective efficiency (%) is indicated. Top, EGFP-U1 70K + mCherry-C1; Middle, EGFP-U1 70K + mCherry-SC35; Bottom, EGFP-U1 70K + mCherry-SC35 in the presence of DRB. Arrowheads indicate high FRET within the nucleoplasm. Bars, 10 μm. (C) FRET efficiencies determined by FLIM for interaction of SC35 with U1 70K and U2AF35 in the presence and absence of DRB. Plot is of mean FRET efficiencies ± SD for 8–11 cells. P-values were obtained as described in A.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p

(A) HeLa cells were cotransfected with EGFP-HCC1 and either mCherry-C1 or mCherry-U2AF35

Confocal images are of transfected cells and FLIM images are of the same cells. The color scale with the respective efficiency (%) is indicated. The FRET efficiencies are shown in continuous pseudocolor. Top, EGFP-HCC1 + mCherry-C1; Middle, EGFP-HCC1 + mCherry-U2AF35; Bottom, EGFP-HCC1 + mCherry-U2AF35 in the presence of DRB. Arrows indicate high FRET within the nucleoplasm and arrowheads indicate nuclear speckles. (B) FRET between HCC1 and U2AF65 measured by FLIM. HeLa cells were transfected with EGFP-HCC1 and cotransfected with either mCherry-C1 or mCherry-U2AF65. Confocal images are of transfected cells and FLIM images are of same cells, in which the percentage of FRET Efficiency and FRET amplitude are shown in pseudocolor. The color scale with the respective efficiency (%) is indicated. Top, EGFP-HCC1 + mCherry-C1; Middle, EGFP-HCC1 + mCherry-U2AF65; Bottom, EGFP-HCC1 + mCherry-U2AF65 in the presence of DRB. Arrowheads indicate high FRET within the nucleoplasm and arrowheads indicate nuclear speckles. Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p

(A) Cell extracts prepared from 293T cells were incubated with either a mouse monoclonal anti–U1 70K antibody bound to Sepharose beads (lanes 2 and 3) or Sepharose beads alone (lanes 4 and 5)

The bound proteins were analyzed by Western blotting with anti-SF2/ASF antibody. Alternatively, the assay was performed in the presence of RNase (lanes 3 and 5). (B) In vivo detection of protein–protein interactions between ECFP-U1 70K and EYFP-SF2/ASF by FRET acceptor photobleaching microscopy. HeLa cells coexpressing ECFP-U1 70K and EYFP-SF2/ASF were analyzed on a wide-field fluorescent microscope. Images were acquired before and after photobleaching. A nonbleached region similar to the bleached region (arrows) was included in the data analysis for comparison. Bars, 15 μm. (C) Donor and acceptor mean fluorescence intensities monitored in the bleached and nonbleached regions were plotted over time. (D) FRET efficiencies for the interaction between ECFP-U1 70K and EYFP-SF2/ASF in the presence and absence of DRB. A FRET efficiency for these interactions was calculated as described in Materials and methods and, when >5%, was considered significant. Plot is of FRET efficiencies ± SD (mean for 8–27 cells) between ECFP + EYFP pairs before and after DRB treatment. P-values were obtained from the two-tailed homoscedastic test comparing the FRET efficiencies with and without DRB treatment.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p

snoMEN knock-down delivered using a lentiviral vector.

<p>(<b>a</b>) Structure of lentiviral vector encoding snoMEN targeted to the endogenous miR21 primary transcript (Lenti-mCherry–pri-miR21 snoMEN). This construct has three snoMEN RNAs (blue pentagons) and an mCherry FP-marker cDNA as described in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138668#pone.0138668.g001" target="_blank">Fig 1A</a></b>, except that the insert was sub-cloned into a vector that would produce the lentivirus particle (pLVX-puro, Clontech). (<b>b</b>) Micrographs show apoptosis induction by transducing with either Lenti-mCherry–pri-miR21-snoMEN, or Lenti-mCherry–Control-snoMEN virus particles. Images were taken 72 hours after transduction. Scale bar is 10 μm. (<b>c</b>) Total RNA from human lung primary (ATCC-CCL-75) and lung Cancer (ATCC-CRL-5868) cells was harvested 24 hours after transduction. Following cDNA synthesis, qPCR was performed using both a matured miR21 specific primer and a universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138668#sec008" target="_blank">methods</a>). GAPDH was used as a control. Graph depicts mean and standard deviation from a minimum of 4 independent experiments. (<b>d</b>) Graph shows a time course of changes in the population of Annexin-V positive cells transduced with either lenti-pri-miR21-snoMEN (Green), lenti-pre-miR21-snoMEN (Blue) or Control (Red). The cell number was counted using FACS. The Annexin-V signal was detected using Guava Nexin Reagent (Guava technologies).</p

(A) HeLa cells were cotransfected with EGFP-U2AF35 and either mCherry-C1 or mCherry-SF2/ASF

Confocal images are of transfected cells and FLIM images of the same cells, in which FRET efficiency and FRET amplitude are shown in pseudocolor. The color scale with the respective efficiency (%) is indicated. Top, EGFP-U2AF35 + mCherry-C1; Middle, EGFP-U2AF35 + mCherry-SF2/ASF; Bottom, EGFP-U2AF35 + mCherry-SF2/ASF in the presence of DRB. Bars, 10 μm. (B) FRET efficiencies determined by FLIM for interaction of SF2/ASF with U2AF35 in the presence and absence of DRB. Plot is of mean FRET efficiencies ± SD for seven to nine cells. To measure the FRET efficiency in the speckles and nucleoplasm, a region characteristic of each was selected for each cell. P-values were obtained as described in the legend. *, P < 0.1.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p