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Double Stranded DNA Breaks and Genome Editing Trigger Ribosome Remodeling and Translational Shutdown

ABSTRACT: Double Stranded DNA Breaks and Genome Editing Trigger Ribosome Remodeling and Translational Shutdown

PROVIDER: PRJNA505780 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8197260.fastq.gz	Fastqsanger.gz
	SRR8197270.fastq.gz	Fastqsanger.gz
	SRR8197261.fastq.gz	Fastqsanger.gz
	SRR8197271.fastq.gz	Fastqsanger.gz
	SRR8197262.fastq.gz	Fastqsanger.gz

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Double Stranded DNA Breaks and Genome Editing Trigger Ribosome Remodeling and Translational Shutdown

Project description:DNA damage activates a robust transcriptional stress response, but much less is known about how DNA damage impacts translation. The advent of genome editing with Cas9 has intensified interest in understanding cellular responses to DNA damage. Here, we find that DNA double-strand breaks (DSBs), including those induced by Cas9, trigger the loss of ribosomal protein RPS27A from ribosomes via p53-independent proteasomal degradation. Comparisons of Cas9 and dCas9 ribosome profiling and mRNA-seq experiments reveal a global translational response to DSBs that precedes changes in transcript abundance. Our results demonstrate that even a single DSB can lead to altered translational output and ribosome remodeling, suggesting caution in interpreting cellular phenotypes measured immediately after genome editing.

2021-11-15 | GSE122615 | GEO

Effect of Oral Administration of Methylene Blue MMX Tablets on Double Stranded DNA

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Transcriptional Stress as a Cause of DNA Double-Stranded Breaks

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APOBEC3s induce mutations during the repair of CRISPR-Cas9-generated DNA breaks.

Project description:APOBEC-AID family of cytidine deaminase prefers single-stranded nucleic acids for cytidine to uracil deamination. Single-stranded nucleic acids are commonly involved in the DNA repair system for breaks generated by CRISPR-Cas9. Here, we show in human cells that APOBEC3s can trigger the cytidine deamination of single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through the homology-directed repair (HDR) of Cas9-generated double-strand breaks . In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks can be further processed to yield mutations mainly involving insertions or deletions (indels). Mechanistically, both APOBEC3-mediated deamination and DNA repair proteins play important roles in the generation of these indels. Correspondingly, optimizing conditions for the repair of CRISPR-Cas9-generated DNA breaks, such as using double-stranded donors in HDR or temporarily suppressing endogenous APOBEC3s, can substantially repress these unwanted mutations in genomic DNA.

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Project description:Goal is to analyze the expression profile of cells shortly after induction of mitochondrial double stranded breaks. ARPE-19 cells are transfected with constructs coding for mitochondrial TALENs, either active or dead. These constructs target two different locations of mtDNA (ATP8 and Dloop). Gene expression is then compared between untransfected and transfected cells (either active or dead TALENs), or between active TALEN (cells with mitochondrial double stranded breaks) and dead TALEN (expressing the same protein without cutting the mitochondrial genome)

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mtDNA breaks compromise mitochondrial membrane ultrastructure and trigger an integrated stress response

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Shutdown is a component of the Drosophila piRNA biogenesis machinery (smallRNA)

Project description:In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping- pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown, a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germ line and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins, with domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones. Though the relevance of these domains to piRNA biogenesis is unknown, evolutionary comparisons raise questions about the integrity of these functions in the shutdown protein. Examination of small RNA levels from nos-GAL4 or tj-GAL4 driven UAS-dsRNA knockdowns of white, shu and piwi in ovaries of Drosophila melanogaster by deep sequencing (using Illumina GAII).

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Strand-specific ChIP-seq at DNA breaks distinguishes single versus doubled stranded DNA binding and refutes single stranded nucleosomes

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Selective ribosome profiling reveals the co-translational chaperone action of trigger factor in vivo

Project description:As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting and folding factors. Here we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone Trigger Factor (TF) reveal that, while TF can interact with many polypeptides, Î²-barrel outer membrane proteins are the most prominent substrates. Loss of TF leads to broad outer membrane defects and premature, cotranslational protein translocation. While in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting ad folding factors. Examination of translation in the Gram-negative bacterium Escherichia coli, as well as an analysis of the interactions between nascent chains and the molecular chaperone Trigger Factor.

2011-12-09 | E-GEOD-33671 | biostudies-arrayexpress

Shutdown is a component of the Drosophila piRNA biogenesis machinery (RNA-seq)

Project description:In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping-pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown, a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germline and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins. Shu contains domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones, though the relevance of these domains to piRNA biogenesis is unknown. Analysis of mRNA expression in Drosophila OSS cells transfected with GFP dsRNA. One sample and replicate, used to establish the OSS baseline transcriptome in the presence of exogenous RNAi activity.

2012-06-30 | E-GEOD-38090 | biostudies-arrayexpress

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