Genomics,Multiomics

Dataset Information

2

Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock


ABSTRACT: 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: Denr knock-down or control NIH3t3 cells were grown to subconfluency in 15 cm plates/replicate. Cells were pre-treated with cycloheximide for 2 min, prior to lysate preparation. Two different sh-RNAs were used for knock-down experiments, and Ribosome profiling (RPF-seq) and parallel RNA-seq libraries were prepared in triplicate for each. For control, non-targeting scramble sh-RNA in triplicate, and Gfp-shRNA in duplicate was used.

INSTRUMENT(S): Illumina HiSeq 2500 (Mus musculus)

ORGANISM(S): Mus musculus  

SUBMITTER: David Gatfield  

PROVIDER: GSE116221 | GEO | 2019-04-01

REPOSITORIES: GEO

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Publications

Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock.

Castelo-Szekely Violeta V   De Matos Mara M   Tusup Marina M   Pascolo Steve S   Ule Jernej J   Gatfield David D  

Nucleic acids research 20190601 10


The non-canonical initiation factor DENR promotes translation reinitiation on mRNAs harbouring upstream open reading frames (uORFs). Moreover, DENR depletion shortens circadian period in mouse fibroblasts, suggesting involvement of uORF usage and reinitiation in clock regulation. 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 uncover  ...[more]

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