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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

Clinically defined SMN mutants show alterations in the association with telomerase complex proteins. (A-B) Cells were transfected with wild-type (WT) or clinically defined SMN mutations, fused to GFP. IPs were performed using anti-GFP antibody. The resulting immunocomplexes were then probed for either dyskerin (A) or NAF1 (B). (C) Graphical representation of the data shown in (A) and (B) (n=8), normalized to the recovery of dyskerin and NAF1 by WT SMN. *P<0.05 compared to WT SMN, which serves as a control. Error bars represent standard error about the mean. For all IPs, inputs represent 1.5% the amount of the lysate used in the IP reaction. Note that GFP vector alone does not recover significant amounts of dyskerin (Fig. 2B, lane 5).
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BIO018804F4: Clinically defined SMN mutants show alterations in the association with telomerase complex proteins. (A-B) Cells were transfected with wild-type (WT) or clinically defined SMN mutations, fused to GFP. IPs were performed using anti-GFP antibody. The resulting immunocomplexes were then probed for either dyskerin (A) or NAF1 (B). (C) Graphical representation of the data shown in (A) and (B) (n=8), normalized to the recovery of dyskerin and NAF1 by WT SMN. *P<0.05 compared to WT SMN, which serves as a control. Error bars represent standard error about the mean. For all IPs, inputs represent 1.5% the amount of the lysate used in the IP reaction. Note that GFP vector alone does not recover significant amounts of dyskerin (Fig. 2B, lane 5).

Mentions: Since we observed, for the first time, that SMN associates with dyskerin and NAF1, we next examined if SMN mutations found in patients with SMA are altered in their ability to interact with these telomerase associated proteins. Four clinically defined SMN mutants were tested (E134K, M263R, Y272C and T274I) (Burghes and Beattie, 2009; Praveen et al., 2014). GFP-tagged wild-type (WT) or mutant SMN plasmids were transfected into HeLa cells, followed 24 h later by lysate generation and IP with anti-GFP antibodies. The amount of dyskerin and NAF1 in the SMN complexes was then examined by western blotting. The background level of dyskerin recovered by the GFP vector alone is shown in Fig. 2B, lane 5. As shown in Fig. 4A and B and quantified in Fig. 4C, the SMN mutants differentially associate with dyskerin (Fig. 4A) and NAF1 (Fig. 4B) relative to the amount of these proteins recovered by WT SMN. Specifically, the E134K mutation decreases the amount of dyskerin in the SMN complex by approximately 50%, relative to WT SMN (Fig. 4A, compare the dyskerin signal in lane 2 to that in lane 1, data quantified in C). In contrast, the M263R and Y272C mutations result in an increase in the amount of NAF1 recovered, but do not differentially affect association with dyskerin. Like the E134K mutation, dyskerin association is decreased in the T274I complex but NAF1 levels are unaffected. These findings indicate that, while all SMN mutants disrupt the normal levels of dyskerin or NAF1 associated with SMN, the mutants can be grouped as dyskerin interaction reducing (E134K/T274I) or NAF1 interaction increasing (M263R/Y272C).Fig. 4.


SMN and coilin negatively regulate dyskerin association with telomerase RNA.

Poole AR, Hebert MD - Biol Open (2016)

Clinically defined SMN mutants show alterations in the association with telomerase complex proteins. (A-B) Cells were transfected with wild-type (WT) or clinically defined SMN mutations, fused to GFP. IPs were performed using anti-GFP antibody. The resulting immunocomplexes were then probed for either dyskerin (A) or NAF1 (B). (C) Graphical representation of the data shown in (A) and (B) (n=8), normalized to the recovery of dyskerin and NAF1 by WT SMN. *P<0.05 compared to WT SMN, which serves as a control. Error bars represent standard error about the mean. For all IPs, inputs represent 1.5% the amount of the lysate used in the IP reaction. Note that GFP vector alone does not recover significant amounts of dyskerin (Fig. 2B, lane 5).
© Copyright Policy - open-access
Related In: Results  -  Collection

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BIO018804F4: Clinically defined SMN mutants show alterations in the association with telomerase complex proteins. (A-B) Cells were transfected with wild-type (WT) or clinically defined SMN mutations, fused to GFP. IPs were performed using anti-GFP antibody. The resulting immunocomplexes were then probed for either dyskerin (A) or NAF1 (B). (C) Graphical representation of the data shown in (A) and (B) (n=8), normalized to the recovery of dyskerin and NAF1 by WT SMN. *P<0.05 compared to WT SMN, which serves as a control. Error bars represent standard error about the mean. For all IPs, inputs represent 1.5% the amount of the lysate used in the IP reaction. Note that GFP vector alone does not recover significant amounts of dyskerin (Fig. 2B, lane 5).
Mentions: Since we observed, for the first time, that SMN associates with dyskerin and NAF1, we next examined if SMN mutations found in patients with SMA are altered in their ability to interact with these telomerase associated proteins. Four clinically defined SMN mutants were tested (E134K, M263R, Y272C and T274I) (Burghes and Beattie, 2009; Praveen et al., 2014). GFP-tagged wild-type (WT) or mutant SMN plasmids were transfected into HeLa cells, followed 24 h later by lysate generation and IP with anti-GFP antibodies. The amount of dyskerin and NAF1 in the SMN complexes was then examined by western blotting. The background level of dyskerin recovered by the GFP vector alone is shown in Fig. 2B, lane 5. As shown in Fig. 4A and B and quantified in Fig. 4C, the SMN mutants differentially associate with dyskerin (Fig. 4A) and NAF1 (Fig. 4B) relative to the amount of these proteins recovered by WT SMN. Specifically, the E134K mutation decreases the amount of dyskerin in the SMN complex by approximately 50%, relative to WT SMN (Fig. 4A, compare the dyskerin signal in lane 2 to that in lane 1, data quantified in C). In contrast, the M263R and Y272C mutations result in an increase in the amount of NAF1 recovered, but do not differentially affect association with dyskerin. Like the E134K mutation, dyskerin association is decreased in the T274I complex but NAF1 levels are unaffected. These findings indicate that, while all SMN mutants disrupt the normal levels of dyskerin or NAF1 associated with SMN, the mutants can be grouped as dyskerin interaction reducing (E134K/T274I) or NAF1 interaction increasing (M263R/Y272C).Fig. 4.

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