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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 as demonstrated by RT-PCR. a) CPEB3 was present in the P60 retina. b) Multiple CPEB3 transcripts were present in the retina. Different primer sets for various CPEB3 transcripts (table1, table 2) were used for PCR. Each visible band was purified and sequenced, which confirmed the presence of six splicing patterns: "+69+24" (lane2, upper band), "-69+24" (lane2, lower band), "exon 11 extension" (lane3), "exon 11 + exon 12" (lane 4, upper band); a new pattern: "exon 11 deletion" (lane 4, lower band), and "partial exon skipping within exon 4". But it did not rule out the presence of "+69-24" and "-69-24", since the competition for the same set of primers by a dominant pattern ("-69+24") may mask the weakly expressed variants. c) Demonstration of "+69-24" and "-69-24" patterns and comparison of tissue distribution of all patterns. Primer sets specific for each individual splicing pattern were used for PCR on thirteen different tissues from adult mice. Each specific band was purified and sequenced for identity confirmation. The results demonstrated the presence of "+69-24" and "-69-24" patterns in the retina (lane 2) in addition to the 6 patterns identified in figure b). It also demonstrated the ubiquity of the majority of the patterns, with the exception of "+69+24", which was expressed in the CNS, the ovary, testis, kidney and heart, but not in the lung, liver, thymus and spleen. d) The locations of these primers were mapped to CPEB3. The corresponding relationships between the numeric primers and the splicing variants in figure a), b) and c) were listed in table 2. Darkened boxed represented exons that could be alternatively spliced.
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Figure 3: The expression of CPEB3 as demonstrated by RT-PCR. a) CPEB3 was present in the P60 retina. b) Multiple CPEB3 transcripts were present in the retina. Different primer sets for various CPEB3 transcripts (table1, table 2) were used for PCR. Each visible band was purified and sequenced, which confirmed the presence of six splicing patterns: "+69+24" (lane2, upper band), "-69+24" (lane2, lower band), "exon 11 extension" (lane3), "exon 11 + exon 12" (lane 4, upper band); a new pattern: "exon 11 deletion" (lane 4, lower band), and "partial exon skipping within exon 4". But it did not rule out the presence of "+69-24" and "-69-24", since the competition for the same set of primers by a dominant pattern ("-69+24") may mask the weakly expressed variants. c) Demonstration of "+69-24" and "-69-24" patterns and comparison of tissue distribution of all patterns. Primer sets specific for each individual splicing pattern were used for PCR on thirteen different tissues from adult mice. Each specific band was purified and sequenced for identity confirmation. The results demonstrated the presence of "+69-24" and "-69-24" patterns in the retina (lane 2) in addition to the 6 patterns identified in figure b). It also demonstrated the ubiquity of the majority of the patterns, with the exception of "+69+24", which was expressed in the CNS, the ovary, testis, kidney and heart, but not in the lung, liver, thymus and spleen. d) The locations of these primers were mapped to CPEB3. The corresponding relationships between the numeric primers and the splicing variants in figure a), b) and c) were listed in table 2. Darkened boxed represented exons that could be alternatively spliced.

Mentions: We next investigated the expression of CPEB3 mRNA within the P60 mouse retina. Regular RT-PCR studies demonstrated that CPEB3 is present in the retina (figure 3a). We further investigated which of the known CPEB3 transcripts identified through our bioinformatics analyses are expressed in the retina. The primer sets for RT-PCR were designed to span exon 5-7, around exon 11, and the partial exon skipping site within exon 4 (figure 3d, table 1, table 2). Since one amplicon may correspond to more than one transcript (figure 1, figure 3d), for our purposes, we named each amplicon as an alternatively spliced "pattern" with a descriptive name rather than a numeric name. For example, the exon 5-7 region has four patterns ("+69+24", "-69+24", "+69-24", and "-69-24"), and the exon 11 region has two patterns ("exon 11 extension", or "exon 11 + exon 12"). The patterns within each splicing region are exclusive of one another. Each pattern correlated to one or multiple transcripts. The correspondence between each pattern and potential CPEB3 transcripts were listed in table 2.


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 as demonstrated by RT-PCR. a) CPEB3 was present in the P60 retina. b) Multiple CPEB3 transcripts were present in the retina. Different primer sets for various CPEB3 transcripts (table1, table 2) were used for PCR. Each visible band was purified and sequenced, which confirmed the presence of six splicing patterns: "+69+24" (lane2, upper band), "-69+24" (lane2, lower band), "exon 11 extension" (lane3), "exon 11 + exon 12" (lane 4, upper band); a new pattern: "exon 11 deletion" (lane 4, lower band), and "partial exon skipping within exon 4". But it did not rule out the presence of "+69-24" and "-69-24", since the competition for the same set of primers by a dominant pattern ("-69+24") may mask the weakly expressed variants. c) Demonstration of "+69-24" and "-69-24" patterns and comparison of tissue distribution of all patterns. Primer sets specific for each individual splicing pattern were used for PCR on thirteen different tissues from adult mice. Each specific band was purified and sequenced for identity confirmation. The results demonstrated the presence of "+69-24" and "-69-24" patterns in the retina (lane 2) in addition to the 6 patterns identified in figure b). It also demonstrated the ubiquity of the majority of the patterns, with the exception of "+69+24", which was expressed in the CNS, the ovary, testis, kidney and heart, but not in the lung, liver, thymus and spleen. d) The locations of these primers were mapped to CPEB3. The corresponding relationships between the numeric primers and the splicing variants in figure a), b) and c) were listed in table 2. Darkened boxed represented exons that could be alternatively spliced.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: The expression of CPEB3 as demonstrated by RT-PCR. a) CPEB3 was present in the P60 retina. b) Multiple CPEB3 transcripts were present in the retina. Different primer sets for various CPEB3 transcripts (table1, table 2) were used for PCR. Each visible band was purified and sequenced, which confirmed the presence of six splicing patterns: "+69+24" (lane2, upper band), "-69+24" (lane2, lower band), "exon 11 extension" (lane3), "exon 11 + exon 12" (lane 4, upper band); a new pattern: "exon 11 deletion" (lane 4, lower band), and "partial exon skipping within exon 4". But it did not rule out the presence of "+69-24" and "-69-24", since the competition for the same set of primers by a dominant pattern ("-69+24") may mask the weakly expressed variants. c) Demonstration of "+69-24" and "-69-24" patterns and comparison of tissue distribution of all patterns. Primer sets specific for each individual splicing pattern were used for PCR on thirteen different tissues from adult mice. Each specific band was purified and sequenced for identity confirmation. The results demonstrated the presence of "+69-24" and "-69-24" patterns in the retina (lane 2) in addition to the 6 patterns identified in figure b). It also demonstrated the ubiquity of the majority of the patterns, with the exception of "+69+24", which was expressed in the CNS, the ovary, testis, kidney and heart, but not in the lung, liver, thymus and spleen. d) The locations of these primers were mapped to CPEB3. The corresponding relationships between the numeric primers and the splicing variants in figure a), b) and c) were listed in table 2. Darkened boxed represented exons that could be alternatively spliced.
Mentions: We next investigated the expression of CPEB3 mRNA within the P60 mouse retina. Regular RT-PCR studies demonstrated that CPEB3 is present in the retina (figure 3a). We further investigated which of the known CPEB3 transcripts identified through our bioinformatics analyses are expressed in the retina. The primer sets for RT-PCR were designed to span exon 5-7, around exon 11, and the partial exon skipping site within exon 4 (figure 3d, table 1, table 2). Since one amplicon may correspond to more than one transcript (figure 1, figure 3d), for our purposes, we named each amplicon as an alternatively spliced "pattern" with a descriptive name rather than a numeric name. For example, the exon 5-7 region has four patterns ("+69+24", "-69+24", "+69-24", and "-69-24"), and the exon 11 region has two patterns ("exon 11 extension", or "exon 11 + exon 12"). The patterns within each splicing region are exclusive of one another. Each pattern correlated to one or multiple transcripts. The correspondence between each pattern and potential CPEB3 transcripts were listed in table 2.

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