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  • In this study we characterized the metabolic function of tra

    2019-10-14

    In this study, we characterized the metabolic function of tra2 in the Drosophila fat body. Similar to the 9G8 phenotype, decreasing tra2 levels in the Drosophila fat body using RNAi resulted in increased starvation resistance and a large increase in triglycerides. This increased storage of triglycerides seemed to be due to an increase in the amount of fat stored per cell and not due to increased food consumption. In addition, the splicing of CPT1 was altered in flies with decreased tra2, resulting in a higher amount of CPT1 isoforms containing exon 6B, which are less catalytically active, potentially explaining the increased triglyceride levels. Together, the results of this study provide in vivo evidence for a connection between tra2-regulated splicing and lipid metabolism.
    Materials and methods
    Results
    Discussion In this study, we set out to determine whether the splicing factor tra2 functions in the Drosophila fat body to regulate lipid metabolism. We have shown that fat body-specific knockdown of tra2 resulted in increased triglyceride storage and starvation resistance. The augmented storage of triglycerides seemed to be due to an increase in the amount of fat stored per cell and not to increased food consumption. Interestingly, we found that the splicing of CPT1 was altered in flies with decreased tra2, resulting in a higher amount of the less active enzyme, potentially explaining the lipid storage phenotype. Previous genome-wide RNAi screens identified a group of genes whose knockdown resulted in smaller, more dispersed droplets [5], [6]. This group included genes such as SmB, SmD and prp8, which are components of various snRNPs in the spliceosome, used for the removal of introns [13]. The effects of altered expression of snRNP proteins on lipid storage in cultured OG-L002 and is consistent with previous studies performed by our lab showing that fat body knockdown of components of the U1 and U2 snRNPs also results in decreased triglyceride levels [5], [6], [7]. Interestingly, altering the expression of SR proteins has different metabolic effects than snRNP gene knockdown as knocking down the SR protein 9G8 in the fat body results in an increase in triglyceride storage [7]. It has also previously been shown that 9G8 binds to the splicing factor tra2 and another RNA-binding protein called transformer (tra) to correctly process the doublesex (dsx) gene, which is important for controlling whether a fly develops into a male or female [9], [14]. Since 9G8 and tra2 act together to regulate sex determination, it is possible that they play similar roles in controlling other processes such as lipid storage and the results described here support that hypothesis. Beyond its function in the splicing of dsx to control sex determination, we have uncovered a new role OG-L002 and for tra2 in the splicing of the gene coding for the metabolic enzyme CPT1. We show here that decreasing tra2 results in the alternative splicing of the gene for CPT1, an enzyme important for lipid breakdown. In tra2 knockdown flies, the inclusion of the alternate sixth exon is altered, resulting in more transcripts that include exon 6B causing the less active CPT1 enzyme to accumulate [8]. This suggests that tra2 may be binding directly to the CPT1 pre-mRNA to regulate the alternative splicing of exons 6A and 6B in the mature CPT1 transcript. This CPT1 splicing phenotype is very similar to the CPT1 splicing defect observed in flies with 9G8 decreased in the fly fat body [7]. Therefore, it is also possible that instead of binding directly to the CPT1 transcript and regulating its alternative splicing, tra2 could form a heterodimer with 9G8 and the entire complex binds to the CPT1 transcript to control its processing. Identifying 9G8 and tra2 binding sequences in the CPT1 pre-mRNA will help clarify these possibilities. In addition to regulating the splicing of CPT1, it is possible that tra2 may regulate the splicing of additional lipid metabolic genes such as fatty acid synthase (dFAS) and acetyl-CoA carboxylase (dACC) which promote the synthesis of fatty acids or brummer (bmm), the fly homolog of adipose triglyceride lipase that is involved in lipolysis [4]. Each of these genes has multiple isoforms, but how the expression of these variants is regulated and whether these different isoforms have different enzyme activities is not known. Experiments designed to measure the splicing of these genes coding for enzymes important for lipid metabolism in tra2-RNAi flies will help provide insight into additional metabolic functions of tra2.