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  • br Materials and methods br Results

    2019-09-10


    Materials and methods
    Results
    Discussion In this study, we have shown that fat body-specific knockdown of the key splicing factors U1-70K and U2AF lead to decreased triglyceride storage phenotypes in larvae. Additionally, there was no accompanying defect in glycogen storage observed in these animals. It is interesting that decreasing the expression of these constitutive splicing factors in the fat body would only lead to an alteration in the splicing of genes involved in triglyceride metabolism and not glycogen as well. This suggests that the activity of lipid metabolic genes are more regulated by changes in the splice variants produced than Naringin clinical involved in glycogen metabolism. Decreased fat body-specific expression of U1-70K and U2AF38 in adult females also yielded a decreased triglyceride level similar to the phenotype observed in larvae. The previous genome-wide RNAi screens have indicated that in vitro knockdown of the constitutive splicing factors U1-snRNA, SmB, SmD, and U2AF50 all result in lipid storage phenotypes [2], [3]. These findings are consistent with our data. We have also shown that knockdown of the SR protein 9G8 in the larval fat body also leads to a decreased triglyceride storage phenotype. Surprisingly, the knockdown of 9G8 in the adult female fat body resulted in a large increase in triglyceride levels and this is mainly due to an increase in the amount of lipid stored in each fat body cell. SR proteins affect splicing of target genes in a concentration-dependent manner, and bind to exonic splice enhancers to promote the use of alternative splice sites [20]. 9G8 expression has been reported to increase in the fat body as the fly transitions through larval and pupal development and has higher expression in the adult female than male (modENCODE data on Flybase), which may provide an explanation for the marked difference between larval and adult triglyceride phenotypes observed here. The elevated triglyceride phenotype observed in flies lacking 9G8 in their fat bodies could occur due to a role for 9G8 in regulating food consumption, lipid synthesis or breakdown, or a combination of these. We have shown that food intake is actually decreased in 9G8-IR flies, which may be a compensatory response to the higher triglycerides. Regardless, changes in feeding do not seem to account for the lipid storage phenotype observed when 9G8 is decreased. Conversely, we have provided evidence that 9G8 affects lipid breakdown by regulating the splicing of CPT1, the rate-determining enzyme in β-oxidation. 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. While we believe that altered splicing of CPT1 contributes to the lipid storage phenotype observed in the 9G8-IR flies, it is probably not the only metabolic gene that is improperly spliced in these flies. A number of important lipid metabolic genes such as dFAS and the adipose triglyceride lipase homolog brummer are annotated to have multiple isoforms (Flybase) and it is possible that 9G8 is involved in controlling the splicing of these genes as well. In fact, the 9G8 splicing enhancer sequence has been identified [21], [22] making it possible to identify metabolic genes in Drosophila that may rely on 9G8 to regulate their alternative splicing. In summary, this study has provided evidence for a role of mRNA splicing in controlling triglyceride storage. We have specifically identified the SR protein 9G8 as a regulator of lipid metabolism by affecting the splicing of the gene encoding for the rate-limiting step in β-oxidation, CPT1, leading to the production of an enzyme with altered kinetics. This study also provides support for using the Drosophila fat body as a system with which to further study the mechanisms of mRNA splicing in an in vivo context.