(A–C) GST-pulldown experiments were conducted using GST, GST-coilin (or fragments thereof) and His-T7-tagged WRAP53 partially purified from bacteria. Pulldown reactions were subjected to SDS-PAGE, Western blotting, and probing with the indicated antibodies. RNase A/T1 treated GST proteins were also used (+). The input lane represents 10% of the His-T7-WRAP53 used in each reaction.

(A) HeLa cells were transfected with control or WRAP53 siRNA. 48 h post transfection, RNA IPs were performed using IgG or α-coilin antibodies, the RNA isolated and analyzed by qRT-PCR. RNA enrichment from the coilin IP was normalized to IgG IP and presented in the histogram. (B) HeLa cells were transfected with control or coilin siRNA. 48 h post transfection, RNA IPs were performed using IgG or α-WRAP53 antibodies, the RNA isolated and analyzed by qRT-PCR. The RNA enrichment from WRAP53 IP was normalized to IgG IP and presented in the histogram. Error bars represent 1 s.d. from 2 experimental repeats and 3 technical repeats; *p<0.005 relative to control KD IP for each RNA.

Schematic of scaRNA 2 and scaRNA 9 showing regions to be amplified by qPCR. HeLa cells were transfected with control, coilin or WRAP53 siRNA. 48 h post transfection, RNA IPs were performed using IgG, α-WRAP53 or α-coilin antibodies, the isolated RNA was converted to cDNA and analyzed by qPCR. The amplicon amounts from WRAP53 IP and coilin IP reactions were normalized (fold change) to that obtained for control IgG IP for each of the siRNA conditions (histogram). Error bars represent 1 s.d. from 3 experimental repeats; *p<0.005 relative to control knockdown (KD) IP for each RNA.

(A) Coilin and GST, purified to homogeneity, were subjected to SDS-PAGE and the gel was Coomassie stained. (B–D) RNA degradation assays using purified coilin or GST, shown in panel A, with 100 nM in vitro transcribed scaRNA 2 (B), scaRNA 9 (C) or scaRNA 9 containing an extended 3′-end (D). RNAs were incubated without protein (0) or with increasing amounts (approximately 0.2 µg, 0.5 µg and 0.7 µg) of GST or coilin. Reactions were then subjected to agarose gel electrophoresis and ethidium bromide staining. The arrowheads in panels B and D demarcate the full-length scaRNA based on expected size.

(A) In vitro transcribed scaRNA 9 with a 3′ extension (100 nM) was incubated with 300 ng of purified GST or coilin (shown in Fig. 4A). Triplicate reactions were conducted. The arrowhead marks the position of the full-length scaRNA 9 with 3′ extension based on expected size. (B) Schematic of scaRNA 9 3′ extension showing regions to be amplified by qRT-PCR. (C) RNA degradation reactions shown in panel A were analyzed by qRT-PCR. The products from the coilin reactions were normalized to that obtained from the GST reactions and presented in the histogram. Error bars represent 1 s.d. from 3 experimental repeats; *p<0.005 relative to product amount in the GST reactions for indicated amplicon.

(A) MEF26 (WT) and MEF42 (coilin knockout) cells were transfected with GFP only or GFP-mouse coilin. Cell lysates were used in TRAP assays, and the products separated by PAGE and visualized by ethidium bromide staining. (B) The TRAP products were quantified by densitometry and normalized to the GFP values for each cell line. Error bars represent 1 s.d. from three separate experiments. In MEF42 cells, the TRAP products are significantly lower in GFP expressing cells compared to GFP-mouse coilin expressing cells (p<0.05).