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ABSTRACT: Drosophila wds RNAi 3rd-instar fat body RNA-seq

PROVIDER: PRJNA894775 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR22099600_1.fastq.gz	Fastqsanger.gz
	SRR22099600_2.fastq.gz	Fastqsanger.gz
	SRR22099601_1.fastq.gz	Fastqsanger.gz
	SRR22099601_2.fastq.gz	Fastqsanger.gz
	SRR22099602_1.fastq.gz	Fastqsanger.gz

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Project description:Purpose: Muscle injury caused by mitocondrial complex I disruption remotedly impairs mitochondrial activity and lipid homeostasis in the fat body . To gain insight into the corss talk between fat body and muscle, we performed a transcriptomic analysis in fat bodies of 3rd instar larvae with complex I-perturbed muscles. Methods: To extract total RNAs for RNA-Seq experiment, we used 10 fat bodies dissected out from both dMef2-Gal4/UAS-white-RNAi (Con) and dMef2-Gal4/UAS-ND-75-RNAi (ND-75-i) 3rd instar larvae. After assessing RNA quality with Agilent Bioanalyzer, mRNAs were enriched by poly-A pull-down. Then, sequencing libraries constructed with Illumina TruSeq RNA prep kit were sequenced using Illumina HiSeq2000 at the Columbia Genome Center (http://systemsbiology.columbia.edu/genome-center). We multiplexed samples in each lane, which yields targeted number of single-end 100 bp reads for each sample, as a fraction of 180 million reads for the whole lane. Sequence reads were Reads were mapped back to fly genome using flybase annotation r5.51 using Tophat with 4 mismatches (--read-mismatches = 4) and 10 maximum multiple hits (--max-multihits = 10). With the uniquely mapped reads, we quantified gene expression levels using Cufflinks (FPKM values) (version 2.0.2) with default settings. Next, we performed data normalization on the read counts and applied a negative binomial statistical framework using the Bioconductor package DESeq to quantify differential expression between experimental and control data. Results: Gene list enrichment analysis of the differentially expressed genes in ND-75-i larval fat body revealed a striking enrichment of multiple metabolic processes impinging on carbohydrate metabolism, amino acid metabolism, and lipid metabolism. However, half of genes that encode mitochondrial proteins are up-regulated. Interestingly, target genes of TGF-beta signaling pathways, including activin signaling and BMP signaling, are enriched in changed transcriptome in ND-75-i larval fat body . In particular, p-Mad and p-dSmad2, are validated with westenr blot or immunostaining. Conclusions: Our study represents that complex I-perturbed muscle remotely decreases mitochondrial activity and subsequent lipid mobilization in the fat body via modulation of TGF-beta signaling. Our results show that RNA-seq offers a comprehensive evaluation of signaling network and biological process in organ communication.

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