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Mus musculus strain:C57BL/6J

ABSTRACT: Transcriptome of mouse liver in Per2::Luc KI and E4bp4 KO / Per2::Luc KI mice

PROVIDER: PRJDB7789 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	DRR188168.fastq.gz	Fastqsanger.gz
	DRR188169.fastq.gz	Fastqsanger.gz
	DRR188170.fastq.gz	Fastqsanger.gz
	DRR188171.fastq.gz	Fastqsanger.gz
	DRR188172.fastq.gz	Fastqsanger.gz

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Project description:Many mammalian proteins have circadian cycles of production and degradation, but de novo transcription is only responsible for a small fraction of this rhythmicity. We used ribosome profiling to quantify RNA translation in liver from circadian-entrained mice transferred to constant darkness conditions over a 24-h period and compared translation levels to absolute protein production for 16 circadian proteins. We observed a delay between translation and peak protein levels for several circadian genes covering a wide range of translation efficiencies. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). Increased uORFs in 5' UTRs was associated with decreased ribosome binding in downstream ORFs and reduced expression of synthetic reporter constructs in vitro and in single cells. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells without altering phase or period. Genomic Per2 uORF mutation using CRISPR/Cas9 increased PER2 expression in PER2:Luc MEFs and reduced sleep in Per2 uORF mutant mice. These results suggest that post-transcriptional processes shape translation of mRNA transcripts, which can impact physiological rhythms and sleep.

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to evaluate the effects of liver-specific E4BP4 overexpression under mouse albumin promoter on the muscle glucose and lipid metabolism. Methods: We generated transgenic mice (TG) with liver-specific E4BP4 overexpression. Gasrocnemius muscles were isolated at Zeitgeber Time (ZT) 0 or 12 from transgenic mice and WT littermates, and total RNA was extracted. Muscle RNA profiles were generated by deep sequencing for four groups with three mouse samples each. Results: There were no significant differences between WT and transgenic mice at both ZT0 and 12. Conclusions: Muscle metabolism was not altered at the transcription level by liver specific overexpression of E4BP4.

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to evaluate the effects of liver-specific E4BP4 overexpression under mouse albumin promoter on the liver glucose and lipid metabolism. Methods: We generated transgenic mice (TG) with liver-specific E4BP4 overexpression, and harvested two livers with one TG mouse each. After cervical dislocation, mouse liver was perfused with a buffer containing Hanks' balanced salt solution, and cross-inked for 30 minutes with 2mM DSG and for 10 minutes with 1% formaldehyde by perfusion, followed by frozen at -80C. Then, Liver was homogenated by gentleMACS Dissociators. Nuclei were isolated and protein-DNA complexes were incubated with antibodies against two kinds of E4BP4 and immunoprecipitated with IgG paramagnetic beads. Results: There were many significant peaks at the promoter/enhancer or intronic region of many genes. Conclusions: E4BP4 possibly regulates the expression of some genes linked to lipid metabolism in the liver.

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