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Next Generation Sequencing Facilitates Quantitative Analysis of Drosophila larval Wild Type and TFAM RNAi (r4-gal4>TFAM RNAi) Fat body Transcriptomes

ABSTRACT: Purpose: The goal of this RNA-Seq is to analyze the transcriptomes from wild type and TFAM RNAi fat body samples at 96 hr AEL (after egg laying) and to identify the differentially expressed genes. Methods: Drosophila larval fat bodies were isolated from wild type and TFAM RNAi (r4-gal4) animals at 96 hrs AEL (after egg laying) of development. mRAN profiles were generated by Next Generation Sequencing Facilities. Results: RNA Seq differential expression analysis identified a significant change in 1758 transcripts, with 1213 transcripts showing reduced expression levels and 545 transcripts with elevated expression levels.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE198604 | GEO | 2022/03/19

REPOSITORIES: GEO

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Project description:The goal of this analysis was to utilize microarray profiling to identify basal alterations in gene expression in response to TFAM depletion and mtDNA stress. Mitochondrial DNA (mtDNA) is normally present at thousands of copies per cell and is packaged into several hundred higher-order structures termed nucleoids. The abundant mtDNA-binding protein, TFAM (transcription factor A,mitochondrial), regulates nucleoid architecture, abundance and segregation. Complete mtDNA depletion profoundly impairs oxidative phosphorylation, triggering calcium-dependent stress signalling and adaptive metabolic responses. However, the cellular responses to mtDNA instability, a physiologically relevant stress observed in many human diseases and ageing, remain poorly defined. Here we show that moderate mtDNA stress elicited by TFAM deficiency engages cytosolic antiviral signalling to enhance the expression of a subset of interferon-stimulated genes. Mechanistically, we find that aberrant mtDNA packaging promotes escape of mtDNA into the cytosol, where it engages the DNA sensor cGAS (also known as MB21D1) and promotes STING (also known as TMEM173)–IRF3-dependent signalling to elevate interferon-stimulated gene expression, potentiate type I interferon responses and confer broad viral resistance. Furthermore, we demonstrate that herpesviruses induce mtDNA stress, which enhances antiviral signalling and type I interferon responses during infection. Our results further demonstrate that mitochondria are central participants in innate immunity, identify mtDNA stress as a cell-intrinsic trigger of antiviral signaling and suggest that cellular monitoring of mtDNA homeostasis cooperates with canonical virus sensing mechanisms to fully engage antiviral innate immunity. Murine embryonic fibroblasts were isolated from wild-type or Tfam+/- E13.5 littermate embryos. RNA from passage-matched wild-type and Tfam+/- MEF lines was extracted in duplicate and hybridized onto Affymetrix microarrays. Four arrays were performed in total with two technical replicates per genotype.

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Next Generation Sequencing Facilitates Quantitative Analysis of Drosophila larval Wild Type and TFAM RNAi (r4-gal4>TFAM RNAi) Fat body Transcriptomes

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