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A small RNA response to DNA ends in Drosophila

ABSTRACT: Small RNAs have been implicated in numerous cellular processes, including effects on chromatin structure and the repression of transposons. We describe the generation of a small RNA response at DNA ends in Drosophila that is analogous to the recently reported DSB-induced RNAs (diRNAs) or Dicer and Drosha dependent small RNAs (ddRNAs) in Arabidopsis and vertebrates. Active transcription in the vicinity of the break amplifies this small RNA response, demonstrating that the normal mRNA contributes to the endo-siRNA precursor. The double-stranded RNA precursor forms with an antisense transcript that initiates at the DNA break. Breaks are thus sites of transcription initiation, a novel aspect of the cellular DSB response. This response is specific to a double-strand break since nicked DNA structures do not trigger small RNA production. The small RNAs are generated independently of the exact end structure (blunt, 3'- or 5'-overhang), can repress homologous sequences in trans and may therefore - in addition to putative roles in repair - exert a quality control function by clearing potentially truncated messages from genes in the vicinity of the break.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE38967 | GEO | 2012/08/03

SECONDARY ACCESSION(S): PRJNA169498

REPOSITORIES: GEO

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A small RNA response to DNA ends in Drosophila

Project description:Small RNAs have been implicated in numerous cellular processes, including effects on chromatin structure and the repression of transposons. We describe the generation of a small RNA response at DNA ends in Drosophila that is analogous to the recently reported DSB-induced RNAs (diRNAs) or Dicer and Drosha dependent small RNAs (ddRNAs) in Arabidopsis and vertebrates. Active transcription in the vicinity of the break amplifies this small RNA response, demonstrating that the normal mRNA contributes to the endo-siRNA precursor. The double-stranded RNA precursor forms with an antisense transcript that initiates at the DNA break. Breaks are thus sites of transcription initiation, a novel aspect of the cellular DSB response. This response is specific to a double-strand break since nicked DNA structures do not trigger small RNA production. The small RNAs are generated independently of the exact end structure (blunt, 3'- or 5'-overhang), can repress homologous sequences in trans and may therefore - in addition to putative roles in repair - exert a quality control function by clearing potentially truncated messages from genes in the vicinity of the break. Drosophila melanogaster S2 cells were cultured and transfected with reporter gene plasmids that were either circular or modified by restriction digest prior to transfection. Following transfection, total RNA was isolated from the cells and gel-purified for size selection (~18-30 nt). Digested plasmid samples were compared to those of circular plasmids and a nontransfected control.

2012-08-03 | E-GEOD-38967 | biostudies-arrayexpress

A Role for Small RNAs in DNA Double-Strand Break Repair

Project description:Here we identify a novel class of small RNAs that are ~21-nucleotide in length and are produced from the sequences in the vicinity of DNA double strand break (DSB) sites in Arabidopsis and humans. We named them diRNAs for DSB-induced small RNAs. In Arabidopsis, the biogenesis of diRNAs requires the PI3 kinase ATR, RNA polymerase IV (Pol IV), and Dicer-like proteins. Mutations in these proteins as well as in Pol V cause significant reduction in DSB repair efficiency. diRNAs are recruited by Argonaute 2 (AGO2) to mediate DSB repair. In humans, knocking down Dicer or Ago2 causes a significant reduction in DSB repair. Our findings reveal a novel biological function for small RNAs in the DSB repair pathway. We propose that diRNAs may function as guide molecules for chromatin modifications or recruitment of repair complexes at DSB sites to facilitate repair. 28 samples from Arabidopsis thaliana in various genetic backgrounds and 5 samples from the human cells, small RNA sequencing

2012-03-26 | E-GEOD-36338 | biostudies-arrayexpress

A Role for Small RNAs in DNA Double-Strand Break Repair

2012-03-27 | GSE36338 | GEO

IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis

Project description:Repair of DNA double-strand break (DSB) is critical for the maintenance of genome integrity. We have previously shown that a class of DSB-induced small RNAs (diRNAs) facilitates homologous recombination (HR)-mediated DSB repair in Arabidopsis thaliana. Here we show that INVOLVED IN DE NOVO 2 (IDN2), a double-stranded RNA (dsRNA) binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA-binding ARGONAUTE 2 (AGO2) leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from ssDNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair.

2017-02-28 | GSE94305 | GEO

Effects of the RNA Exosome on damage induced small RNAs in the 28S ribosomal locus

Project description:The repair of DNA double strand breaks (DSBs) has recently been shown to depend not only on protein function but also on RNA. In particular, the processing of a long damage-induced RNA into small damage induced RNAs (diRNAs) has been a focus of interest. Given that the RNA exosome has been shown in Drosophila and human cells to participate in DSB repair, we investigated whether the catalytic components EXOSC10 and DIS3 have a function in diRNA biogenesis in human cells.

2019-04-10 | GSE113109 | GEO

Next-generation sequencing reveals two populations of damage-induced small RNAs at endogenous DNA double-strand breaks

Project description:Recent studies suggest that the repair of DNA double-strand breaks (DSBs) is supported by damage-induced small RNAs (diRNAs) that are generated by Drosha and Dicer from transcripts that originate from the regions flanking the break. However, transcription and diRNA production at naturally occurring DSBs in mammalian genomes remain controversial. We have used the homing endonuclease I-PpoI to produce DSBs in human and mouse cells, and we have analyzed the biogenesis of diRNAs at I-PpoI induced breaks in the 28S rDNA and the Ryr2 gene. We provide direct evidence that RNA polymerase II transcribes the sequences that flank the DSBs. Our results also reveal that the resulting transcripts are processed into two types of diRNAs with characteristic length and terminal nucleotide signatures. Furthermore, diRNA production is independent of Drosha and DGCR8, and only a subset of diRNAs depends on Dicer, as shown by small RNA analyses of knock-out cells that lack specific RNA processing enzymes. Our findings are compatible with previous observations of a general transcription inhibition at DSBs and provide novel insights into the mechanisms of RNA synthesis and processing at DSBs.

2018-10-22 | GSE106764 | GEO

ANKRD31 protein interactions

Project description:Sex chromosomes in males of most mammalian species share only a small homologous segment, the pseudoautosomal region (PAR), wherein double-strand break (DSB) formation must occur for correct meiotic segregation. We find that multiple DSB-promoting factors hyperaccumulate in the PAR, including the ankyrin-repeat domain-containing protein ANKRD31. We used immunoprecipitation and mass spectrometry from extracts of juvenile mouse testes to identify potential ANKRD31-interacting proteins.

2020-06-19 | PXD017191 | Pride

Z-DNA underlies the target choice of Aire by facilitating promoter poising [BLISS]

Project description:We profiled DNA double-strand break (DSB) formation genome-wide in medullary thymic epithelial cells to investigate how DSBs regulate Aire's target choices

2024-03-11 | GSE224552 | GEO

NEDD8 S65 phosphorylation under different stress conditions

Project description:In this project we present the identification of NEDD8 S65 phosphorylation under mitochondrial and DNA double strand break (DSB) stress conditions. Relative comparison revealed elevated levels of pNEDD8 under mitochondrial stress and reduced levels of pNEDD8 under DSB stress.

2021-11-03 | PXD027477 | Pride

Comparative proteomic analysis identifies differentially expressed proteins associated with meiotic arrest in cattle-yak hybrids

Project description:Comparative proteomic analysis identified a total of 452 differentially abundant proteins (DAPs) in the fluorescence-activated cell sorting (FACS) isolated spermatocytes from 12-month-old yak and cattleyak. 291 proteins were only identified in yak spermatocytes. Gene Ontology analysis revealed that the downregulated DAPs were mostly enriched in the cellular response to DNA damage stimulus and double-strand break (DSB) repair via break-induced replication, while the proteins specific for yak were related to cell division and cycle, spermatogenesis, and negative regulation of the extrinsic apoptotic signaling pathway.

2023-03-21 | PXD043039 | iProX

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