Alternative Exon Usage in the Single CPT1 Gene of Drosophila Generates Functional Diversity in the Kinetic Properties of the Enzyme

Abstract

The Drosophila melanogaster genome contains only one CPT1 gene (Jackson, V. N., Cameron, J. M., Zammit, V. A., and Price, N. T. (1999) Biochem. J. 341, 483-489). We have now extended our original observation to all insect genomes that have been sequenced, suggesting that a single CPT1 gene is a universal feature of insect genomes. We hypothesized that insects may be able to generate kinetically distinct variants by alternative splicing of their single CPT1 gene. Analysis of the insect genomes revealed that (a) the single CPT1 gene in each and every insect genome contains two alternative exons and (ii) in all cases, the putative alternative splicing site occurs within a small region corresponding to 21 amino acid residues that are known to be essential for the binding of substrates and of malonyl-CoA in mammalian CPT1A. We performed PCR analyses of mRNA from different Drosophila tissues; both of the anticipated splice variants of CPT1 mRNA were found to be expressed in all of the tissues tested (both in larvae and adults), with the expression level for one of the splice variants being significantly different between flight muscle and the fat body of adult Drosophila. Heterologous expression of the full-length cDNAs corresponding to the two putative variants of Drosophila CPT1 in the yeast Pichia pastoris revealed two important differences between the properties of the two variants: (i) their affinity (K 0.5 ) for one of the substrates, palmitoyl-CoA, differed by 5-fold, and (ii) the sensitivity to inhibition by malonyl-CoA at fixed, higher palmitoyl-CoA concentrations was 2-fold different and associated with different kinetics of inhibition. These data indicate that alternative splicing that specifically affects a structurally crucial region of the protein is an important mechanism through which functional diversity of CPT1 kinetics is generated from the single gene that occurs in insects.Lipid metabolism in insects has largely been investigated from the perspective of energy supply to flight muscle, particularly in migratory flight. Because their lipid metabolism is less complex than in vertebrates, insects are considered a good model system in which to study the fundamental aspects of fat metabolism. The increasing availability of insect gene sequence data and completely sequenced genomes allows comparative analyses with mammalian data and suggests that many of the fundamental molecular mechanisms involved in fatty acid metabolism and their regulation are conserved. Although in most insects there are major differences in the glycerolipids used to transport lipid between tissues (diacylglycerol in insects versus triacylglycerol in vertebrates), the transfer of the long chain acyl moiety across the mitochondrial inner membrane is likely to be substantially similar to that in vertebrates and to involve the carnitine shuttle (1). We have previously shown that the fruit fly Drosophila melanogaster has only a single CPT1 (carnitine palmitoyltransferase 1) gene (2). When we expressed the full-length cDNA (Berkeley Dro...

The control of lipid metabolism by mRNA splicing in Drosophila

“…The Drosophila genome contains one homolog of CPT1 , the rate-limiting step in β-oxidation. This gene has two distinct isoforms resulting from an alternate sixth exon (exon 6A or 6B; [15]). Enzymes resulting from CPT1 isoforms that include exon 6A have higher activity than those including exon 6B [15].…”

“…This gene has two distinct isoforms resulting from an alternate sixth exon (exon 6A or 6B; [15]). Enzymes resulting from CPT1 isoforms that include exon 6A have higher activity than those including exon 6B [15]. To determine whether 9G8 affects triglyceride storage by regulating the splicing of CPT1 , we performed quantitative PCR for the presence of these two alternative sixth exons of CPT1 in 9G8 knockdown fat bodies.…”

“…9G8-IR flies express twice the amount of CPT1 mRNA including exon 6B than CPT1 mRNA including exon 6A leading to twice as much of the less active CPT1 enzyme (Fig. 4; [15]). Therefore, it is likely that flies with decreased 9G8 in their fat body break down triglycerides through β-oxidation more slowly resulting in the accumulation of triglyceride shortly after eclosion.…”

“…Remnant genomic DNA was removed using the DNA free kit (Ambion) and cDNA was generated from 1 μg of total RNA using oligo (dT) as a primer using the RETROscript kit (Ambion) according to manufacturer’s instructions. Quantitative PCR for CPT1 isoforms was carried out as previously described [15]. …”

Expression of spliced variants ofperiodmRNA in the Japanese honeybeeApis cerana japonica

“…These data indicate that per a and b are variants produced by alternative splicing of this GT-AG intron (Shimizu et al 2001). mRNA variants generated by alternative splicing of other single genes code functionally different proteins in insects (Price et al 2010). The secondary structures of the per proteins encoded by the a/b transcripts were estimated by the New Joint Method Noguchi 1991, Ito et al 1997) using the Parallel Protein Information Analysis system (http://mbs.cbrc.jp/papia/ papia.html).…”