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Developmental expression and phylogenetic conservation of alternatively spliced forms of the C-terminal binding protein corepressor - PubMed

Developmental expression and phylogenetic conservation of alternatively spliced forms of the C-terminal binding protein corepressor

Priya Mani-Telang et al. Dev Genes Evol. 2007 Feb.

Abstract

The C-terminal binding protein (CtBP) is an evolutionarily conserved transcriptional corepressor found in multicellular eukaryotes. Multiple forms of the protein are typically found in animal cells, produced from separate genes and by alternative splicing. CtBP isoforms have also been implicated in cytoplasmic functions, including Golgi fission and vesicular trafficking. All forms of CtBP contain a conserved core domain that is homologous to alpha-hydroxyacid dehydrogenases, and a subset of isoforms (CtBP(L)) contain extensions at the C terminus. Despite distinct developmental profiles and knockout phenotypes in the mouse, the properties of different isoforms of the protein are found to be similar in many transcriptional assays. We have investigated the expression and conservation of distinct isoforms of the CtBP protein in insects and found that the expression of multiple, developmentally regulated isoforms is widely conserved. In a variety of Drosophila species, the relative abundance of CtBP(L) to CtBP(S) drops sharply after embryogenesis, revealing a conserved developmental shift. Despite the overall lower levels of this isoform, bioinformatic analysis reveals that exons encoding the C-terminal extension in CtBP(L) are conserved from Diptera to Coleoptera, suggesting that the CtBP(L) isoform contributes an important, evolutionarily conserved function.

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Figures

Fig. 1
Fig. 1. Developmental expression profile of CtBP isoforms in Drosophila melanogaster

A.Specificity of α-CtBP antibody tested in Western blot with Drosophila melanogaster embryonic extract (lanes 1,2) or bacterial extracts containing recombinant CtBPL (lane 3) or CtBPS (lane 4). Preimmune serum did not cross react with any proteins in embryo extract, while α-CtBP recognized two isoforms of approximately 42 and 50 kDa in embryonic extracts. Recombinant proteins migrate slower than endogenous counterparts due the presence of an N-terminal hexahistidine tag and a C-terminal Flag tag. Markers (kDa) are indicated to the left. B. Expression of CtBP isoforms in embryos, larvae, pupae, and adults. 50 μg of total soluble protein was loaded on 10% SDS-PAGE and analyzed by immunoblotting with anti-CtBP. Relative CtBPL and CtBPS levels were unchanged during embryogenesis. A marked reduction in the relative level of CtBPL was observed from the larval through adult stages. CtBPs levels remained relatively unchanged throughout the developmental time course. The bottom panel shows β-tubulin as a loading control. C. Steady-state levels of CtBP mRNAs measured by RT-PCR analysis. Total mRNA from embryos and adults was reverse transcribed and PCR amplified using primers specific to CtBPL exons, CtBPS regions, or a region common to both isoforms as indicated. Reverse transcription reactions were primed with 60, 30, or 15 ng of total RNA, as indicated by triangular symbol. The –RT control reactions were primed with 60 ng of RNA. Based on quantitation of biological replicates, the ratio of CtBPL to CtBPS products was measured to be approximately 1:1 in adults compared to 4:1 in embryos.

Fig. 2
Fig. 2. Conservation of coding information for CtBPL-specific C-terminus

A. Peptide coding information present in dipterans, bee, and beetle genomic sequences homologous to alternatively spliced exon 6 and 7 in Drosophila melanogaster encoding CtBP “tail” region. Conceptual translations of genomic sequences are shown below sequence of CtBPL, in which YPEG represents the end of the exon 5 coding sequence for the CtBPL isoform. Predicted intron size in nucleotides is indicated between exons. The introns in Apis mellifera have apparently been eliminated. Dark gray (purple) shading indicates widely conserved sequences; light gray (yellow) shading partially conserved sequences. Possible sumoylation sites (I/V K X E) are indicated by gray (red) bars above exon 7 residues. An alternative splice acceptor site 5’ of the junction shown for exon 7 would produce an mRNA encoding an additional VSSQS motif at the beginning of exon 7; this sequence is not conserved outside of Drosophila, unlike the case shown in 2B. B. The cDNA sequences reported for D. melanogaster CtBPL contain alternative splice acceptor sites for the 5’ end of exon 6; a sequence isolated from adult head uses a downstream acceptor site (NP_001014617), while a different sequence isolated from embryo uses a more upstream acceptor ((Sutrias-Grau and Arnosti, 2004)) incorporating the residues LNGGYYT. This portion of the protein is evolutionarily conserved and contains appropriate splice acceptor sequences both 5’ and 3’ of region, thus alternative splicing may be a conserved feature here as well.

Fig. 3
Fig. 3. Conserved developmental regulation of CtBP protein expression in D. mojavensis and D. virilis

Expression of CtBP isoforms in embryos, larvae, pupae, and adults of D. mojavensis (A.) and D. virilis (B.). As in D. melanogaster, two predominant species were observed in both species, but the CtBPL isoform has a lower mobility (~60kDa vs. 50kDa in D. melanogaster). The relative levels of CtBPL to CtBPS in the embryo was greater in these species than in D. melanogaster, but just as in that species there is a pronounced decrease in relative levels of CtBPL in the larva and pupa. Adult levels of CtBPL remain low in D. mojavensis, but recover in D. virilis. 50 μg of total soluble protein was loaded on 10% SDS-PAGE and analyzed by immunoblotting with anti-CtBP.

Fig. 4
Fig. 4. Adult expression of CtBP proteins in four Drosophila species, Anopheles gambiae, Apis mellifera, and Tribolium casteneum

Soluble extracts from adults were analyzed by Western blotting using anti-CtBP. Cross-reacting species similar in size to CtBPS were noted in all Dipterans. Slower mobility proteins consistent with CtBPL-like species were present in all extracts; multiple bands were detected in extracts from all species except Tribolium. The relative abundance of CtBPL and CtBPS is masked by the long exposure of the gel; lower panel shows a separate Western blot (lanes 8-11) that was exposed for a shorter time to demonstrate the lower abundance of CtBPL to CtBPS in D. melanogaster, D. sechellia, and D. mojavensis adults.

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