The regulation of lipid storage by the splicing factor transformer2 (tra2) in Drosophila

Open Access
Author:
Mikoluk, Cezary
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Justin DiAngelo, Thesis Supervisor
  • Sandy Feinstein, Honors Advisor
Keywords:
  • tra
  • tra2
  • transformer
  • transformer2
  • drosophila
  • melanogaster
  • CPT1
  • triglyceride
  • lipid
  • storage
  • splicing
  • factor
  • 9G8
Abstract:
Excess nutrients are stored as triglycerides mainly in the adipose tissue of an animal. These triglycerides are located in structures called lipid droplets within adipose cells. Previous genome-wide RNAi screens in Drosophila cells identified splicing factors as playing a role in lipid droplet formation. Dr. DiAngelo identified the SR protein, 9G8, as an important factor in fat storage as decreasing its levels results in augmented triglyceride storage in the fat body. However, whether 9G8 interacts with other proteins to affect lipid metabolism is unclear. Previous in vitro studies have implicated 9G8 in the control of sex determination by binding to transformer (tra) and transformer2 (tra2) to regulate doublesex (dsx) splicing; any function of these proteins in regulating metabolism is unknown. The goal of this study is to determine whether tra2 regulates fat storage in vivo. To test this hypothesis, we measured triglyceride and glycogen levels in flies with tra2dsRNA induced in the adult fat body. Decreasing the expression of this sex determination gene resulted in an increase in triglyceride levels but no effect on glycogen storage, a phenotype similar to the 9G8 knockdown flies. Consistent with the triglyceride phenotypes, tra2 knockdown flies lived longer under starvation conditions. In addition, this increase in triglycerides is due to more storage of these molecules per cell and not an increase in the number of fat cells as DNA levels are unchanged in tra2dsRNA flies compared to controls. This triglyceride accumulation phenotype also does not seem to be due to more feeding as food consumption was not increased in the tra2dsRNA flies as measured by CAFÉ assay. We next wanted to determine whether the nutrient storage phenotype observed here is due to altered expression of the genes coding for important metabolic enzymes. While the levels of major lipid metabolic enzymes were mostly unchanged, the splicing of CPT1, an enzyme involved in the breakdown of lipids, was altered in flies with decreased tra2. The less-catalytically active isoform of CPT1 which accumulated in tra2dsRNA flies suggests a decrease in lipid breakdown, which is consistent with the increased triglyceride levels observed in these flies. Together, these results suggest a link between mRNA splicing, sex determination and lipid metabolism and may provide insight into the mechanisms underlying tissue-specific splicing and nutrient storage in the fat body.