Limits...
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

Mapping of the interaction domain on coilin for association with the dyskerin complex. (A) Schematic representation of coilin showing the locations of the self-association domain (SA Domain), RNA binding domain (RBD), RG-Box, and TUDOR-like domain (TUDOR) of the various GFP-tagged coilin constructs. (B) Cells were transfected with empty GFP vector or GFP-tagged coilin constructs, followed by IP with antibodies to GFP. Probing antibodies are indicated. For the GFP signals, the same membrane probed with anti-dyskerin was also probed with polyclonal anti-GFP antibodies. The bands for each GFP-tagged protein were then grouped together (lower panel). The experiment was repeated four times, and a representative result is shown. For all IPs, inputs represent 1.5% of cell lysate used in the IP reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4920197&req=5

BIO018804F2: Mapping of the interaction domain on coilin for association with the dyskerin complex. (A) Schematic representation of coilin showing the locations of the self-association domain (SA Domain), RNA binding domain (RBD), RG-Box, and TUDOR-like domain (TUDOR) of the various GFP-tagged coilin constructs. (B) Cells were transfected with empty GFP vector or GFP-tagged coilin constructs, followed by IP with antibodies to GFP. Probing antibodies are indicated. For the GFP signals, the same membrane probed with anti-dyskerin was also probed with polyclonal anti-GFP antibodies. The bands for each GFP-tagged protein were then grouped together (lower panel). The experiment was repeated four times, and a representative result is shown. For all IPs, inputs represent 1.5% of cell lysate used in the IP reaction.

Mentions: We next set out to delineate the region of coilin that is necessary for incorporation into the telomerase complex. For these studies, we utilized coilin fragments fused at the N-terminus to GFP (Fig. 2A). Human coilin is 576 amino acids in length and contains a self-association domain (Hebert and Matera, 2000), an RNA binding/processing domain (Broome and Hebert, 2013), an RG box (Hebert et al., 2001) and a Tudor-like domain (Shanbhag et al., 2010). Cells were transfected with empty GFP vector, plasmids encoding GFP-tagged full-length coilin or fragments thereof. After 24 h, lysates from these cells were subjected to IP with anti-GFP antibodies. As shown in Fig. 2B, GFP-coilin recovers the most amount of dyskerin relative to that recovered by GFP alone. Deletion of the RNA binding/processing domain (RBD, Δ121-291) greatly decreases the amount of dyskerin recovered, but this region alone (GFP-121-291F) is not necessary and sufficient for dyskerin association. These findings demonstrate that coilin association with dyskerin, and by extension, telomerase, requires the RBD of coilin, but amino acids upstream and downstream of the RBD region must facilitate this interaction.Fig. 2.


SMN and coilin negatively regulate dyskerin association with telomerase RNA.

Poole AR, Hebert MD - Biol Open (2016)

Mapping of the interaction domain on coilin for association with the dyskerin complex. (A) Schematic representation of coilin showing the locations of the self-association domain (SA Domain), RNA binding domain (RBD), RG-Box, and TUDOR-like domain (TUDOR) of the various GFP-tagged coilin constructs. (B) Cells were transfected with empty GFP vector or GFP-tagged coilin constructs, followed by IP with antibodies to GFP. Probing antibodies are indicated. For the GFP signals, the same membrane probed with anti-dyskerin was also probed with polyclonal anti-GFP antibodies. The bands for each GFP-tagged protein were then grouped together (lower panel). The experiment was repeated four times, and a representative result is shown. For all IPs, inputs represent 1.5% of cell lysate used in the IP reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920197&req=5

BIO018804F2: Mapping of the interaction domain on coilin for association with the dyskerin complex. (A) Schematic representation of coilin showing the locations of the self-association domain (SA Domain), RNA binding domain (RBD), RG-Box, and TUDOR-like domain (TUDOR) of the various GFP-tagged coilin constructs. (B) Cells were transfected with empty GFP vector or GFP-tagged coilin constructs, followed by IP with antibodies to GFP. Probing antibodies are indicated. For the GFP signals, the same membrane probed with anti-dyskerin was also probed with polyclonal anti-GFP antibodies. The bands for each GFP-tagged protein were then grouped together (lower panel). The experiment was repeated four times, and a representative result is shown. For all IPs, inputs represent 1.5% of cell lysate used in the IP reaction.
Mentions: We next set out to delineate the region of coilin that is necessary for incorporation into the telomerase complex. For these studies, we utilized coilin fragments fused at the N-terminus to GFP (Fig. 2A). Human coilin is 576 amino acids in length and contains a self-association domain (Hebert and Matera, 2000), an RNA binding/processing domain (Broome and Hebert, 2013), an RG box (Hebert et al., 2001) and a Tudor-like domain (Shanbhag et al., 2010). Cells were transfected with empty GFP vector, plasmids encoding GFP-tagged full-length coilin or fragments thereof. After 24 h, lysates from these cells were subjected to IP with anti-GFP antibodies. As shown in Fig. 2B, GFP-coilin recovers the most amount of dyskerin relative to that recovered by GFP alone. Deletion of the RNA binding/processing domain (RBD, Δ121-291) greatly decreases the amount of dyskerin recovered, but this region alone (GFP-121-291F) is not necessary and sufficient for dyskerin association. These findings demonstrate that coilin association with dyskerin, and by extension, telomerase, requires the RBD of coilin, but amino acids upstream and downstream of the RBD region must facilitate this interaction.Fig. 2.

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