GSE124790
Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock
The non-canonical initiation factor DENR promotes translation reinitiation on uORF-containing mRNAs. Moreover, DENR depletion shortens circadian period in mouse fibroblasts, suggesting that uORF usage and reinitiation regulate clock function. To identify DENR-regulated translation events transcriptome-wide and, in particular, specific core clock transcripts affected by this mechanism, we have used ribosome profiling in DENR-deficient NIH3T3 cells. We found 240 transcripts with altered translation rate, and used linear regression analysis to extract uORF features predictive of DENR dependance. Among core clock genes, we identified Clock as a DENR target. Using Clock 5'UTR mutants, we mapped the specific uORF through which DENR acts to regulate CLOCK protein biosynthesis. Notably, these experiments identified an alternative downstream start codon, which likely represents the true CLOCK N-terminus. Our findings provide insights into uORF-mediated translational regulation that can regulate the mammalian circadian clock and gene expression at large. Overall design: iCLIP experiments: Denr-overexpressing (OE) or non-transfected (NT) NIH3t3 cells were grown to subconfluency in 15 cm plates/replicate. Cells were crosslinked with UV-C (254 nm) at 150 mJ/cm2,prior to lysate preparation. RNA was partially digested, and DENR-RNA complexes were pulled down with anti-DENR antibody (pre-immobilised in protein G dynabeads). An infrarred adaptor was ligated in the 3' end of RNAs and complexes were visualised after PAGE and nitrocellulose transfer in a Licor machine. RNA-protein complexes of 60-130 Kda were excised and used for library preparation. 24 PCR cycles were used for final library amplification. An IgG immunoprecipitation was used as IP control.