Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA279465 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR1930190.fastq.gz | Fastqsanger.gz | |||
| SRR1930200.fastq.gz | Fastqsanger.gz | |||
| SRR1930210.fastq.gz | Fastqsanger.gz | |||
| SRR1930220.fastq.gz | Fastqsanger.gz | |||
| SRR1930230.fastq.gz | Fastqsanger.gz |
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Project description:Ribosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in wide-spread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods. Ranging concentrations of cycloheximide and various stress contitions were tested with Ribo-seq
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Project description:In this study, we generated nucleotide-resolution translatome as well as transcriptome data of isolated primary cardiomyocytes undergoing hypertrophy. More than 10,000 open reading frames (ORF) were detected from the deep sequencing of ribosome protected fragments orchestrating the shift of translatome in hypertrophied cardiomyocytes.We further identified more than 100 potential micropeptides encoded by uncharacterized small open reading frames (sORFs) in the long noncoding RNA genes. Over two-thirds of these micropeptide candidates were experimentally validated in a random test with three micropeptides showing regulatory function in cardiomyocyte hypertrophy via modulating the activities of Oxidative phosphorylation, Calcium signaling pathway, and MAPK signaling pathway.Our study provided a genome-wide overview of the translational controls of cardiomyocyte hypertrophy and demonstrated an unrecognized role of micropeptides in cardiomyocyte biology.
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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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