Fig. 5.

Reduction of Cajal body proteins alters the composition of the dyskerin complex. (A, upper panel) siRNA-treated lysate was IPed with anti-dyskerin. A portion of the IPs were subjected to SDS-PAGE, western blotting, and probing with anti-SMN or anti-dyskerin antibodies, while another portion of the IPs was used to isolate RNA for the experiment shown in C. (Lower panel) Three different siRNAs (Coil2, Coilin 3′ UTR or CoilA) were used to knockdown coilin in HeLa cells. Cells were also transfected with control siRNA. 48 h later, lysates were generated and IPed using a dyskerin monoclonal antibody. The IPs were then subjected to SDS-PAGE and western blotting. The membrane was then probed with a SMN antibody. Also shown is the knockdown of WRAP53 we achieve using WRAP53 siRNA compared to control, coilin or SMN siRNA. Western blot was probed with anti-WRAP53 then anti-tubulin antibodies. (B) siRNA-transfected cell lysate was IPed with anti-dyskerin antibody. The IPs were subjected to SDS-PAGE, western blotting and probing with anti-coilin antibodies. The location of full-length coilin and the 28 kDa coilin derivative is indicated. For all IPs, inputs represent 1.5% the amount of the lysate used in the IP reaction. (C) Coilin and SMN negatively regulate hTR association with dyskerin. siRNA-transfected lysate was IPed with dyskerin polyclonal antibody, followed by RNA isolation and qRT-PCR to determine the level of hTR, normalized to that obtained with control siRNA (P-values are shown, n=4 experimental sets). Error bars represent standard error about the mean.