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SMN and coilin negatively regulate dyskerin association with telomerase RNA.

Poole AR, Hebert MD - Biol Open (2016)

Bottom Line: Telomerase is a ribonucleoprotein comprising telomerase RNA and associated proteins.Here we demonstrate newly discovered associations between SMN and coilin with telomerase components, and further show that reduction of SMN or coilin is correlated with increased association of telomerase RNA with one these components, dyskerin.Collectively, our findings bolster the link between SMN, coilin and the coilin derivative in the biogenesis of telomerase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Mississippi Medical Center, Jackson, MS 39216-4505, USA.

No MeSH data available.


Related in: MedlinePlus

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.
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BIO018804F5: 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.

Mentions: Because SMN, WRAP53 and coilin have all been shown to associate with each other (Enwerem et al., 2014; Hebert et al., 2001; Mahmoudi et al., 2010), and interact with telomerase proteins (Bachand et al., 2002; Tycowski et al., 2009; Venteicher et al., 2009) (Figs 1–4), we next tested the effect of the reduction of one of these proteins on the association of the other proteins with dyskerin. For these studies, cells were treated with control, coilin, WRAP53 or SMN siRNAs for 48 h, followed by lysate generation and IP with anti-dyskerin antibodies. The siRNAs used here reduce protein levels by at least 80% (Fig. 5A, upper panel, lane 4 and lower right panel, lane 3; Fig. 5B, lane 2) (Enwerem et al., 2014). Interestingly, coilin reduction results in a significant increase (1.5-fold, n=5, P<0.005) in the amount of SMN recovered in the dyskerin complex (Fig. 5A). This increase of SMN in the dyskerin complex upon coilin reduction was also observed when using two additional coilin siRNAs (Fig. 5A, lower left panel). In contrast, the reduction of WRAP53 did not alter the amount of SMN associated with dyskerin (Fig. 5A), nor did this treatment change the amount of coilin recovered by dyskerin IP (Fig. 5B). Furthermore, SMN knockdown (KD) did not alter the amount of coilin associated with dyskerin (Fig. 5B).Fig. 5.


SMN and coilin negatively regulate dyskerin association with telomerase RNA.

Poole AR, Hebert MD - Biol Open (2016)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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BIO018804F5: 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.
Mentions: Because SMN, WRAP53 and coilin have all been shown to associate with each other (Enwerem et al., 2014; Hebert et al., 2001; Mahmoudi et al., 2010), and interact with telomerase proteins (Bachand et al., 2002; Tycowski et al., 2009; Venteicher et al., 2009) (Figs 1–4), we next tested the effect of the reduction of one of these proteins on the association of the other proteins with dyskerin. For these studies, cells were treated with control, coilin, WRAP53 or SMN siRNAs for 48 h, followed by lysate generation and IP with anti-dyskerin antibodies. The siRNAs used here reduce protein levels by at least 80% (Fig. 5A, upper panel, lane 4 and lower right panel, lane 3; Fig. 5B, lane 2) (Enwerem et al., 2014). Interestingly, coilin reduction results in a significant increase (1.5-fold, n=5, P<0.005) in the amount of SMN recovered in the dyskerin complex (Fig. 5A). This increase of SMN in the dyskerin complex upon coilin reduction was also observed when using two additional coilin siRNAs (Fig. 5A, lower left panel). In contrast, the reduction of WRAP53 did not alter the amount of SMN associated with dyskerin (Fig. 5A), nor did this treatment change the amount of coilin recovered by dyskerin IP (Fig. 5B). Furthermore, SMN knockdown (KD) did not alter the amount of coilin associated with dyskerin (Fig. 5B).Fig. 5.

Bottom Line: Telomerase is a ribonucleoprotein comprising telomerase RNA and associated proteins.Here we demonstrate newly discovered associations between SMN and coilin with telomerase components, and further show that reduction of SMN or coilin is correlated with increased association of telomerase RNA with one these components, dyskerin.Collectively, our findings bolster the link between SMN, coilin and the coilin derivative in the biogenesis of telomerase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Mississippi Medical Center, Jackson, MS 39216-4505, USA.

No MeSH data available.


Related in: MedlinePlus