Coilin knockdown results in accumulation of primary U snRNA transcripts.

<p><i>A</i>, relative U snRNA levels in HeLa cells following coilin knockdown. Error bars represent 1 standard deviation of fold change, n = 9. Statistical analysis performed using a paired Student's Ttest of the change in Ct relative to GAPDH between control and coilin knockdown RNA. * denotes p<0.04. <i>C</i>, diagrams of U snRNA genes with locations of primers used for qRT-PCR analysis noted.</p

Protein constructs and purification to homogeneity by electro-elution.

<p><i>A</i>, Schematic of full length human coilin with line diagrams of bacterially expressed constructs below. RG box denotes the 4 Arg-Gly repeat at amino acids 413–420. Construct names are listed to the left and theoretical iso-electric points with and without GST tag are to the right of each construct line diagram. Diamonds represent mutations to Asp or Glu, mimicking phosphorylation by addition of negative charge. Circles represent mutations of the 4 Arg in the RG box to Gly. <i>B</i>, Coomassie stained SDS PAGE with samples from bacterially expressed proteins purified to homogeneity by electro-elution. <i>C</i>, Coomassie stained SDS PAGE with samples from a control BL-21 purification or bacterially expressed fly coilin protein purified to homogeneity by electro-elution.</p

Purified coilin binds DNA.

<p>All reactions contain purified electro-eluted protein and 13 ng DNA. After incubation, reactions were loaded into 1% agarose gels containing ethidium bromide. The location of the approximately 3000 bp unbound DNA is indicated. Lane 1 for each gel represents control reactions without protein. <i>A</i>, negative control GST; Lane 2, 0.22 µg; Lane 3, 2.2 µg. <i>B</i>, negative control GST-pirin; Lane 2, 0.2 µg; Lane 3, 2 µg. <i>C</i>, coilin wt; Lane 2, 0.13 µg; Lane 3, 0.5 µg. <i>D</i>, RNase treated coilin wt; Lanes 2–6, 0.1, 0.2, 0.4, 0.6, 0.8 µg. <i>E</i>, negative control GST, 0.29 µg; coilin construct GST-N362, 0.29 µg; coilin P construct, 0.29 µg. <i>F</i>, GST-C214, GST-C214 P and GST-C214mtRG; Lanes 2–3, 0.29 and 0.57 µg; Lanes 4–5, 0.29 and 0.57 µg; Lanes 6–7, 0.2 and 2 µg. <i>G</i>, <i>D. melanogaster</i> coilin either untreated or pre-treated with RNase A/T1; Lane 2, 0.19 µg untreated protein; Lane 3, 0.19 µg RNase treated; Lane 4, 0.19 untreated protein alone; line is drawn to highlight the slight DNA mobility shift. Arrows in <i>C</i>, <i>D</i>, and <i>G</i> indicate the location of a distinct protein/DNA complex, resulting in slower migration than unbound DNA. Asterisk (*) under lanes denotes protein amount included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036300#pone-0036300-t001" target="_blank">Table 1</a> for DNA binding.</p

Purified coilin cleaves U2 RNA transcript.

<p><i>A</i>, Diagram of U2 gene repeat region including snRNA coding region and extending ∼630 bp beyond. Primers used for qRT-PCR are denoted; forward primers above and reverse primers below diagram. <i>B</i>, Diagram of protocol for incubations and subsequent analysis of U2 RNA via qRT-PCR. <i>C</i>, graph of relative U2 RNA qRT-PCR product amount following incubation with purified electro-eluted coilin. Values represent fold change of product levels following coilin incubation, normalized to GST incubation set at 1. Error bars represent 1 standard deviation of fold change, n = 9. Statistical analysis performed using a paired Student's Ttest of GST incubated and coilin incubated Ct values. * denotes p<0.03, ** denotes p<0.0005.</p

Purified coilin has RNase activity in its N terminal region.

<p>All reactions, unless indicated, contain either 25 or 100 ng purified electro-eluted protein (left to right) and 500 ng HeLa RNA. After incubation, reactions were loaded into 1% agarose gels containing ethidium bromide. 28S and 18S ribosomal RNA bands are denoted. A control reaction containing RNA but not protein is shown in lane 1 of each panel. Negative control proteins are GST and GST-pirin. <i>A</i>, negative controls GST and GST-pirin. <i>B</i>, coilin wt. <i>C</i>, negative control, GST; coilin constructs GST-N362 and coilin P. <i>D</i>, coilin C terminal constructs. <i>E</i>, <i>D. melanogaster</i> (fly) coilin. <i>F</i>, incubations of 250 ng coilin wt and/or RNase A/T1 cocktail (2.5 pg RNase A and 0.075 pg RNase T1) with 500 ng HeLa RNA with or without 0.1 units RNase inhibitor; + or − signs denote presence or absence of the component listed to the left in each reaction.</p

Summary of RNase and nucleic acid binding activity of purified proteins.

a<p>Protein amounts determined by comparative densitometric analysis of Coomassie stained bands in SDS-PAGE gel to a known protein gradient. Nucleic acid amounts determined by spectrophotometric analysis.</p>b<p>“−” denotes little or no activity up to 0.1 µg protein; “+” denotes clearly visible RNA degradation at 0.1 µg protein; “+ +” denotes the most RNA degradation of all proteins.</p>c<p>“none” denotes that with the highest protein amount tested (in brackets), none of the DNA was visibly shifted; “Partial” denotes either the DNA band was visibly more diffuse as compared to the control lanes, or that a distinct band was seen in reactions with protein migrating slower than the unbound DNA; “Complete” denotes that in reactions with the specified amount of protein, no band was seen migrating at the location of the band in the control reaction.</p>d<p>Volumes listed are those seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036300#pone-0036300-g001" target="_blank">Figure 1</a> (6 L fly coilin culture was used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036300#pone-0036300-g001" target="_blank">Figure 1</a>). For GST, the latter volume was used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036300#pone.0036300.s003" target="_blank">Figure S3</a>.</p

Coilin purified by electro-elution contains DNA and RNA.

<p><i>A</i>, 500 ng electro-eluted coilin wt; arrows 1–5 denote distinct nucleic acid species. <i>B</i>, 500 ng electro-eluted coilin wt either DNase I or RNase A/T1 treated for 30 m at 37°C. DNase treated lane contains only RNA species indicated by arrows 3 and 4. RNase treated lanes contains only DNA species indicated by arrows 1 and 5. <i>C</i>, 500 ng electro-eluted coilin wt after 10 days at 4°C.</p

Schematic representation of human coilin showing the locations of the coilin self-interaction domain and apparent nucleolar localization signal (NoLS) [<b>18</b>], nuclear localization signals (NLS), RG box [<b>21</b>] and tudor domain [<b>19</b>].

<p>Also indicated are 11 residues that are phosphorylated. Blue represents phosphorylation sites enriched during mitosis, yellow corresponds to those identified during interphase, and the phosphoresidues identified in both mitosis and interphase are green <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025743#pone.0025743-Toyota1" target="_blank">[9]</a>.</p

Reduction of coilin, WRAP53 or SMN dysregulates processing of ectopically expressed scaRNA2.

<p>(A) Schematic showing ectopic pre-processed full-length scaRNA2 (expressed from the pcDNA3.1+ vector) and the locations of the U2-25 domain and the processed guide RNA (mgU2-61). A poly A tail, which does not exist for endogenous scaRNA2, is indicated. The probe used for Northern blots is also shown. (B) Northern blot of HeLa RNA (20 μg/lane) from either non-transfected cells (lane 2) or cells transfected (24 hr) with pcDNA 3.1+ scaRNA2 (lane 3). The probe used is shown in (A) The lower panel shows a longer exposure of the region of the gel with mgU2-61 signal in order to better visualize the endogenous signal. (C) Northern blot of Hela RNA (10 μg/lane) from cells transfected with control (lane 2), coilin (lane 3), WRAP53 (lane 4) or SMN (lane 5) siRNA for 24 hrs then transfected with pcDNA 3.1+ scaRNA2 for 24 hrs. The probe used is shown in (A). Ectopic pre-processed full-length (denoted as A), ectopic processed full-length (denoted as B) and endogenous full-length scaRNA2 is shown. An arrowhead marks an intermediate species often seen in RNA isolated from coilin knockdown cells (lane 3). (D) Histogram quantifying the ratio between ectopic processed full-length and ectopic pre-processed full-length (B/A) from 8 different experimental sets. Standard deviation was used to include error bars. Students t test was used to determine statistical significance, indicated by “*” and corresponding to a p value less than 0.05. (E) Northern blot of Hela RNA (20 μg/lane) from cells transfected with control (lane 1), coilin (lane 2), WRAP53 (lane 3) or SMN (lane 4) siRNA for 24 hrs then transfected with pcDNA 3.1+ scaRNA2 for 24 hrs. The Northern blotting transfer conditions were optimized to retain the smaller, mgU2-61 guide RNA. The probe used is shown in (A). The majority of the signal is coming from ectopically expressed mgU2-61 guide RNA. (F) Histogram quantifying the mgU2-61 guide RNA from 8 different experimental sets. The mobility of a DIG-labeled RNA marker (in nucleotides) is shown. Standard deviation was used to include error bars. Students t test was used to determine statistical significance, indicated by “*” and corresponding to a p value less than 0.05.</p

Co-expression of SMN and coilin in the WI-38 primary cell line promotes foci formation.

<p>(A) WI-38 cells were co-transfected with myc-tagged SMN and coilin, followed by fixation and detection of the expressed proteins using anti-SMN or anti-coilin antibodies and appropriate secondary antibodies. DAPI was used to stain the nucleus. In the merged image, the nucleus is blue, SMN is green and coilin signal is red. Foci with co-localized SMN and coilin are yellow. (B) WI-38 cells were co-transfected with myc-SMN and GFP-WRAP53, followed by fixation and detection of the expressed proteins. Anti-SMN antibody was used to detect SMN and the nucleus was stained with DAPI. In the merged image, SMN is red, GFP-WRAP53 is green and the nucleus is blue.</p