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Characterization of the transcripts and protein isoforms for cytoplasmic polyadenylation element binding protein-3 (CPEB3) in the mouse retina.

Wang XP, Cooper NG - BMC Mol. Biol. (2009)

Bottom Line: The relative abundance of the patterns in the retina is demonstrated.The level of CPEB3 was up-regulated in the retina during development.The presence of multiple CPEB3 isoforms indicates remarkable complexity in the regulation and function of CPEB3.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomical Sciences and Neurobiology, Health Sciences Campus, 500 S, Preston Street, University of Louisville, Louisville, KY, USA. x0wang04@gwise.louisville.edu

ABSTRACT

Background: Cytoplasmic polyadenylation element binding proteins (CPEBs) regulate translation by binding to regulatory motifs of defined mRNA targets. This translational mechanism has been shown to play a critical role in oocyte maturation, early development, and memory formation in the hippocampus. Little is known about the presence or functions of CPEBs in the retina. The purpose of the current study is to investigate the alternative splicing isoforms of a particular CPEB, CPEB3, based on current databases, and to characterize the expression of CPEB3 in the retina.

Results: In this study, we have characterized CPEB3, whose putative role is to regulate the translation of GluR2 mRNA. We identify the presence of multiple alternative splicing isoforms of CPEB3 transcripts and proteins in the current databases. We report the presence of eight alternative splicing patterns of CPEB3, including a novel one, in the mouse retina. All but one of the patterns appear to be ubiquitous in 13 types of tissue examined. The relative abundance of the patterns in the retina is demonstrated. Experimentally, we show that CPEB3 expression is increased in a time-dependent manner during the course of postnatal development, and CPEB3 is localized mostly in the inner retina, including retinal ganglion cells.

Conclusion: The level of CPEB3 was up-regulated in the retina during development. The presence of multiple CPEB3 isoforms indicates remarkable complexity in the regulation and function of CPEB3.

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Related in: MedlinePlus

The expression of CPEB3 proteins in the retina. a) The regulation of CPEB3 protein during the postnatal development of the retina. CPEB3 western blot on retinal samples from seven different postnatal ages showed that the 75 kD band protein was increased during the development. Another band at ~130 kD was also present in the retina. Both bands diminished when the antibody was pre-adsorbed with the recombinant CPEB3 protein (the last lane). GAPDH was used as a loading control. b) Both of the 75 kD and the 130 kD bands diminished when Hela cells were transfected with CPEB3 siRNA. Cells in the negative control were treated with a negative control siRNA. GAPDH was used as a loading control.
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Figure 6: The expression of CPEB3 proteins in the retina. a) The regulation of CPEB3 protein during the postnatal development of the retina. CPEB3 western blot on retinal samples from seven different postnatal ages showed that the 75 kD band protein was increased during the development. Another band at ~130 kD was also present in the retina. Both bands diminished when the antibody was pre-adsorbed with the recombinant CPEB3 protein (the last lane). GAPDH was used as a loading control. b) Both of the 75 kD and the 130 kD bands diminished when Hela cells were transfected with CPEB3 siRNA. Cells in the negative control were treated with a negative control siRNA. GAPDH was used as a loading control.

Mentions: We investigated the relative abundance of CPEB3 protein in the developing retina with the aid of western blots. Two antibody-labeled bands with molecular weights of ~130 kD and ~75 kD respectively were identified in western blots and were both diminished by pre-adsorption with recombinant CPEB3 protein or by depletion of CPEB3 in Hela cells (figure 6). The ~75 kD band could be isoform 3 or isoform 4 based on their predicted sizes. The intensity of the ~75 kD band increased significantly during postnatal development of the retina, which was consistent with the postnatal increases in the level of CPEB3 transcripts (figure 4, 5). The ~130 kD band could be a dimer, a pre-protein, or a prion form of CPEB3. Since the ~130 kD band was not weakened in reducing and denaturing conditions (by adding 1,4-dithioerythritol or beta-mercaptoethanol and boiling), it seems less likely to be a dimer. The possibility of it being a pre-protein needs further evidence. The transcripts shown here do not indicate the presence of a protein larger than ~78 kD, neither is there any such protein evident in the UniProt database. However, a previous study indicated the presence of a ~100 kD CPEB3 protein [10]. With the aid of a different gel/buffer system and different protein standards, we have since found that the larger band appeared to be around 97 kD. Therefore, it seems likely that the 100 kD band in the prior study and the 130 kD band in our study are of the same identity. We can only speculate that any transcripts for CPEB3 proteins larger than 78 kD are yet to be discovered. The possibility of it being a prion form [32,33] also needs further investigation.


Characterization of the transcripts and protein isoforms for cytoplasmic polyadenylation element binding protein-3 (CPEB3) in the mouse retina.

Wang XP, Cooper NG - BMC Mol. Biol. (2009)

The expression of CPEB3 proteins in the retina. a) The regulation of CPEB3 protein during the postnatal development of the retina. CPEB3 western blot on retinal samples from seven different postnatal ages showed that the 75 kD band protein was increased during the development. Another band at ~130 kD was also present in the retina. Both bands diminished when the antibody was pre-adsorbed with the recombinant CPEB3 protein (the last lane). GAPDH was used as a loading control. b) Both of the 75 kD and the 130 kD bands diminished when Hela cells were transfected with CPEB3 siRNA. Cells in the negative control were treated with a negative control siRNA. GAPDH was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807433&req=5

Figure 6: The expression of CPEB3 proteins in the retina. a) The regulation of CPEB3 protein during the postnatal development of the retina. CPEB3 western blot on retinal samples from seven different postnatal ages showed that the 75 kD band protein was increased during the development. Another band at ~130 kD was also present in the retina. Both bands diminished when the antibody was pre-adsorbed with the recombinant CPEB3 protein (the last lane). GAPDH was used as a loading control. b) Both of the 75 kD and the 130 kD bands diminished when Hela cells were transfected with CPEB3 siRNA. Cells in the negative control were treated with a negative control siRNA. GAPDH was used as a loading control.
Mentions: We investigated the relative abundance of CPEB3 protein in the developing retina with the aid of western blots. Two antibody-labeled bands with molecular weights of ~130 kD and ~75 kD respectively were identified in western blots and were both diminished by pre-adsorption with recombinant CPEB3 protein or by depletion of CPEB3 in Hela cells (figure 6). The ~75 kD band could be isoform 3 or isoform 4 based on their predicted sizes. The intensity of the ~75 kD band increased significantly during postnatal development of the retina, which was consistent with the postnatal increases in the level of CPEB3 transcripts (figure 4, 5). The ~130 kD band could be a dimer, a pre-protein, or a prion form of CPEB3. Since the ~130 kD band was not weakened in reducing and denaturing conditions (by adding 1,4-dithioerythritol or beta-mercaptoethanol and boiling), it seems less likely to be a dimer. The possibility of it being a pre-protein needs further evidence. The transcripts shown here do not indicate the presence of a protein larger than ~78 kD, neither is there any such protein evident in the UniProt database. However, a previous study indicated the presence of a ~100 kD CPEB3 protein [10]. With the aid of a different gel/buffer system and different protein standards, we have since found that the larger band appeared to be around 97 kD. Therefore, it seems likely that the 100 kD band in the prior study and the 130 kD band in our study are of the same identity. We can only speculate that any transcripts for CPEB3 proteins larger than 78 kD are yet to be discovered. The possibility of it being a prion form [32,33] also needs further investigation.

Bottom Line: The relative abundance of the patterns in the retina is demonstrated.The level of CPEB3 was up-regulated in the retina during development.The presence of multiple CPEB3 isoforms indicates remarkable complexity in the regulation and function of CPEB3.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomical Sciences and Neurobiology, Health Sciences Campus, 500 S, Preston Street, University of Louisville, Louisville, KY, USA. x0wang04@gwise.louisville.edu

ABSTRACT

Background: Cytoplasmic polyadenylation element binding proteins (CPEBs) regulate translation by binding to regulatory motifs of defined mRNA targets. This translational mechanism has been shown to play a critical role in oocyte maturation, early development, and memory formation in the hippocampus. Little is known about the presence or functions of CPEBs in the retina. The purpose of the current study is to investigate the alternative splicing isoforms of a particular CPEB, CPEB3, based on current databases, and to characterize the expression of CPEB3 in the retina.

Results: In this study, we have characterized CPEB3, whose putative role is to regulate the translation of GluR2 mRNA. We identify the presence of multiple alternative splicing isoforms of CPEB3 transcripts and proteins in the current databases. We report the presence of eight alternative splicing patterns of CPEB3, including a novel one, in the mouse retina. All but one of the patterns appear to be ubiquitous in 13 types of tissue examined. The relative abundance of the patterns in the retina is demonstrated. Experimentally, we show that CPEB3 expression is increased in a time-dependent manner during the course of postnatal development, and CPEB3 is localized mostly in the inner retina, including retinal ganglion cells.

Conclusion: The level of CPEB3 was up-regulated in the retina during development. The presence of multiple CPEB3 isoforms indicates remarkable complexity in the regulation and function of CPEB3.

Show MeSH
Related in: MedlinePlus