In prior work, we showed that Dis3 and Rrp6 physic ally interact and co localize in S2 cells and are mutually required for proper localization. To determine whether these protein partners co localize and cooperate in flies, we stained WT fly brains with antibodies to Inhibitors,Modulators,Libraries Rrp6 and Dis3. Surprisingly, anti Rrp6 antibodies do not stain the brain lobes, whereas anti Dis3 antibodies do, anti Rrp6 antibodies stain certain brainstem portions, but this staining is not found in all brain stains. Further, Dis3 depletion did not significantly affect the anti Rrp6 antibody staining pattern. These observations suggest that Dis3 and Rrp6 may not cooperate in all Drosophila tissues, consistent with the exozyme hypothesis.

Transcriptomic profiling of Dis3 knock down flies Given the role of Dis3 in regulating a defined Inhibitors,Modulators,Libraries subset of the S2 cell transcriptome, we hypothesized that Dis3 depletion affects fly development by perturbing either the expression, processing, and or turnover of vital de velopmental transcripts. To test this hypothesis in an unbiased and thorough manner, we performed RNA deep sequencing analysis of WT and Dis3KD flies during development. To capture snapshots of the fly transcriptome at specific developmental stages, we divided our analysis into 6 time points. At the first time point, embryos were collected after flies laid eggs for 18 hours. For all other time points, the flies laid eggs for 4 hours and samples were collected after 26, 50, 74, 98 and 122 hours. We collected WT and Dis3KD flies in parallel to permit comparison.

Following RNA extraction, purification, Brefeldin_A preparation, and deep sequencing, the raw RNA seq data was processed, quantified, and normalized, and RPKM values were calculated. From this analysis, a total of 14,623 transcripts were mapped to the Drosophila gen ome, including 19 new, previously unannotated genes. Of these transcripts, the 11,665 that had high raw read count in at least one sample were selected for fur ther analysis. To organize those transcripts, we gener ated a heatmap with the log2 transformed RPKM values for every time point. Our heatmap revealed a specific RNA accumulation pattern in day 0 and day 4 Dis3KD samples as compared to the WT samples. We isolated this tran script subset and generated a detailed heatmap. To determine the nature of these effects, we performed a gene ontology en richment study.

In Inhibitors,Modulators,Libraries three Inhibitors,Modulators,Libraries GO categories�� biological process, cellular components, and molecular func tion ��we selected the five GO terms with the top P value scores and then graphed them by both number of transcripts and fold enrichment. The highest scoring GO terms in the Dis3KD data set correspond to biometabolism of metabolites, chemical energy, mitochondria, and membrane transporters. Notably, these GO terms are unified in the phenomenon of oxidative phosphorylation, suggest ing that Dis3 may be involved in this process.