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Ribosome profiling of 293T cells with or without miRNA inhibitors or non-cleaving shRNAs

ABSTRACT: Ribosome profiling of 293T cells with or without miRNA inhibitors or non-cleaving shRNAs

PROVIDER: PRJNA861947 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR20613391.fastq.gz	Fastqsanger.gz
	SRR20613392.fastq.gz	Fastqsanger.gz
	SRR20613393.fastq.gz	Fastqsanger.gz
	SRR20613394.fastq.gz	Fastqsanger.gz
	SRR20613395.fastq.gz	Fastqsanger.gz

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Ribosome profiling of 293T cells with or without miRNA inhibitors or non-cleaving shRNAs

Project description:Cells treated with NC control, miRNA inhibitors (miR-361-3p, 375-3p, 769-5p, and 4521), Scr control, or non-cleaving shNBD1s were harvested for ribosome profiling

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Ribosome profiling and mRNA-seq of muscle skeletal muscle and DROSHA KO 293T cells

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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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Project description:Ribosome profiling with translation inhibitors reveals pervasive translation in murine ES cells. Ribosome profiling or stranded RNAseq (ribominus) with murine embryonic stem cells treated with either DMD-pateamineA, Puromycin, Harringtonine or vehicle (no drug control). Two replicates per condition.

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Ribosome profiling with translation inhibitors reveals pervasive translation in murine ES cells

Project description:Ribosome profiling with translation inhibitors reveals pervasive translation in murine ES cells.

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Translation Inhibitors Cause Abnormalities in Ribosome Profiling Experiments

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Ribosome profiling and mRNA-seq of muscle skeletal muscle and DROSHA KO 293T cells

Project description:Ribosome profiling and mRNA-seq of muscle skeletal muscle and DROSHA KO 293T cells

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Human Embryonic Kidney(293T) Cells: Control vs. Transfected

Project description:Transcriptional profiling of Human Embryonic Kidney(293T) Cells comparing control untreated 293T cells with 293T cells transfected with A) pcDNA 3.0(-) vector[Invitrogen] (Mock) and B) Expression vector pcDNA 3.0(-) containing cloned Influenza virus H5N1and H11N1-NS1 (Non-Structural1) gene.

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DNA methylation profiling for HEK-293T cells transfected with GFP and DMEΔ

Project description:Methylome data obtained from human embryonic kindey (HEK)-293T cells expressing a GFP (293T-GFP) or a truncated form of Arabidopsis DEMETER (DME) 5-methylcytosine (5mC) DNA glycosylase (293T-DMEΔ) analyzed on a Human Methylation 450K BeadChip platform (Illumina). These methylation array data revealed genome-wide DNA methylation patterns of the 293T-GFP cells (without direct 5mC excision activity) and 293T-DMEΔ cells (with artificially implemented direct 5mC excision activity).

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Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors

Project description:Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation is regulated differently from canonical translation is poorly understood. We thus used start codon-selective reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of non-AUG reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA are resistant to cycloheximide (CHX), a translation inhibitor which slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes cause queuing of scanning pre-initiation complexes (PIC), preferentially enhancing otherwise poor recognition of non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. Moreover, PIC queuing can cause translation from an AUG codon in a poor context to become less sensitive to CHX. We further find that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence, but not those targeting newly initiated ribosomes. In total, these data indicate that ribosome queuing enables mRNAs with poor initiation context, namely those from non-AUG start codons, to be resistant to pharmacological inhibitors.

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