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Using ribosome profiling to investigate the effect of p53 on translation in cancer cells

ABSTRACT: Using ribosome profiling to investigate the effect of p53 on translation in cancer cells

PROVIDER: PRJEB33244 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR3403470.fastq.gz	Fastqsanger.gz
	ERR3403480.fastq.gz	Fastqsanger.gz
	ERR3403490.fastq.gz	Fastqsanger.gz
	ERR3403500.fastq.gz	Fastqsanger.gz
	ERR3403471.fastq.gz	Fastqsanger.gz

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Using ribosome profiling to investigate the effect of p53 on translation in cancer cells

Project description:p53 is deleted or dysfunctional, or develops these features during the course of disease, with a frequency of about 50% in human cancers. This is clinically important because this group of patients respond very poorly to treatment. Our approach to discovering novel biomarkers or targets for therapy for p53 deleted/dysfunctional disease is to find genes that are translationally regulated by p53. The aim of this experiment is to compare translational efficiencies in cancer cells that are p53 WT, p53-/-, or p53 R175H, in response to doxorubicin.

2021-02-22 | E-MTAB-8100 | biostudies-arrayexpress

Genome-Wide Analysis In Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling

Project description:Techniques for systematically monitoring protein translation have lagged far behind methods for measuring mRNA levels. Here we present a ribosome profiling strategy, based on deep sequencing of ribosome protected mRNA fragments, that enables genome-wide investigation of translation with sub-codon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control both for determining absolute protein abundance and for responding to environmental stress. We also observed distinct phases during translation involving a large decrease in ribosome density going from early to late peptide elongation as well as wide-spread, regulated initiation at non-AUG codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible. Examine replicates of ribosome footprints and mRNA abundance in biological replicates of log-phase growth and acute amino acid starvation

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Using mitoribosomal profiling to investigate human mitochondrial translation

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Translation Inhibitors Cause Abnormalities in Ribosome Profiling Experiments

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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