Drosha drives the formation of DNA:RNA hybrids around DNA break sites to facilitate DNA repair

Lu, Wei-Ting; Hawley, Ben R; Skalka, George L.; Baldock, Robert A; Smith, Ewan M.; Bader, Aldo S.; Malewicz, Michal; Watts, Felicity Z.; Wilczynska, Ania

doi:10.1038/s41467-018-02893-x

Cited by 186 publications

(269 citation statements)

References 74 publications

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“…We speculate that the presence of a GC-rich sequence downstream of the TOP motif in RP genes (Meyuhas and Kahan, 2015) may explain in part why R-loops formed at RPG loci are stable and capable of reducing the rate of transcription, unless they are actively destabilized by the Ddx5 helicase. It has been reported that the Microprocessor complex is recruited to DNA double strand break (DSB) sites, where it facilitates R-loop formation and promotes DSB repair (Bonath et al, 2018;Crossley et al, 2019;Lu et al, 2018). Thus, the Microprocessor may participate in both the formation and the resolution of R-loops, depending on the cellular context.…”

Section: Discussionmentioning

confidence: 99%

Control of ribosomal protein synthesis by Microprocessor complex

Jiang

Prabhakar

Voorn

et al. 2020

Preprint

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Ribosome biogenesis in eukaryotes requires stoichiometric production and assembly of 80 ribosomal proteins (RPs) and 4 ribosomal RNAs, and its rate must be coordinated with cellular growth. The indispensable regulator of RP biosynthesis is the 5'-terminal oligopyrimidine (TOP) motif, spanning the transcription start site of all RP genes. Here we show that the Microprocessor complex, previously linked to the first step of processing microRNAs (miRNAs), coregulates RP expression by binding the TOP motif of nascent RP mRNAs and stimulating transcription elongation via resolution of DNA/RNA hybrids. Cell growth arrest triggers nuclear export and degradation of the Microprocessor protein Drosha by the E3 ubiquitin ligase Nedd4, accumulation of DNA/RNA hybrids at RP gene loci, decreased RP synthesis, and ribosome deficiency, hence synchronizing ribosome production with cell growth. Conditional deletion of Drosha in erythroid progenitors phenocopies human ribosomopathies, in which ribosomal insufficiency leads to anemia. Outlining a miRNA-independent role of the Microprocessor complex at the interphase between cell growth and ribosome biogenesis offers a new paradigm by which cells alter their protein biosynthetic capacity and cellular metabolism.in response to the change in cellular growth environment remains to be identified (Meyuhas and Kahan, 2015; Patursky-Polischuk et al., 2014).During transcription, a three-stranded nucleic acid structure known as an R-loop spontaneously forms(García-Muse and Aguilera, 2019). It is composed of a DNA/RNA hybrid (a single-stranded template DNA (ssDNA) hybridized with a nascent mRNA) and an associated non-template ssDNA (García-Muse and Aguilera, 2019;Sanz and Chédin, 2019;Sanz et al., 2016). R-loops are critical modulator of gene expression and DNA repair (García-Muse and Aguilera, 2019;Sanz and Chédin, 2019;Sanz et al., 2016), and their extended persistence during transcription is inhibitory to gene expression (Gowrishankar et al., 2013;Nudler, 2012). The abundance of R-loops is determined by the balance between its formation and resolution of DNA/RNA hybrids and various factors, including transcription factors, helicases, ribonucleases, topoisomerases, chromatin remodelers, proteins in DNA repair and RNA surveillance, have been identified to control R-loop homeostasis(García-Muse and Aguilera, 2019). Deregulation of R-loops, which results in aberrant gene expression and chromatin structure, increased DNA breaks, and genome instability, contributes to human disease, including neurological disorders and tumorigenesis (García-Muse and Aguilera, 2019;Groh and Gromak, 2014;Sanz et al., 2016).The Microprocessor complex comprises two core components, the ribonuclease (RNase) III Drosha and its cofactor Dgcr8. It is essential for the biogenesis of most microRNAs (miRNAs), short RNAs that repress gene expression by binding to messenger RNAs and targeting them for degradation and/or preventing their translation (Han et al., 2004;Siomi and Siomi, 2010). The Microprocessor localizes predom...

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Section: Discussionmentioning

confidence: 99%

Control of ribosomal protein synthesis by Microprocessor complex

Jiang

Prabhakar

Voorn

et al. 2020

Preprint

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“…The degree of chromatin compaction is thought to influence repair pathway choice. DSBs occurring in open chromatin undergo end resection and are predominantly repaired by HR, which results in faithful repair and suppresses dangerous mutations from arising in coding regions of the genome (5,7,18). The preference for employing HR to repair DSBs in transcribed regions is not due to cell cycle-dependent availability of factors.…”

Section: Discussionmentioning

confidence: 99%

Chromatin-associated MRN complex protects highly transcribing genes from genomic instability

Salifou

Burnard

Basavarajaiah

et al. 2020

Preprint

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The MRN-MDC1 complex plays a central role in the DNA damage response (DDR) and repair.Using Proteomics of Isolated Chromatin Fragments (PICh), we identified DDR factors, such as MDC1, among those that become highly associated with a genomic locus upon transcriptional activation. Purification of endogenous MDC1, in the absence of exogenous DNA damage, revealed its interaction with factors involved in gene expression and co-transcriptional RNA processing, in addition to DDR factors. ChIP-seq analysis showed that MDC1 interacting factors, MRE11 and NBS1 subunits of MRN, were co-localized throughout the genome and notably at TSSs and gene bodies of actively transcribing genes. Blockade of transcriptional elongation showed that binding of MRN was dependent on the RNAPII transcriptional complex rather than transcription per se. Depletion of MRN increased RNAPII abundance at TSSs and gene bodies of MRE11/NBS1-bound genes. Prolonged exposure of cells to either MRE11-or NBS1-depletion led to single nucleotide polymorphism formation across actively transcribing, MRE11 or NBS1 target genes. These data support a model by which association of the MRN complex with the transcriptional machinery constitutively scans active genes for transcription-induced DNA damage to preserve the integrity of the coding genome.

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“…Single‐gene studies and a genome wide transcriptional analysis of DSBs induced at AsiSI sites could not identify dilncRNAs originating from transcriptionally inactive regions. [ 28,29 ] However, case studies of specific, unique intergenic DSBs as well as an in vitro model of linearized transcriptionally inactive plasmids that mimics DSBs, could detect dilncRNAs even from silent loci. [ 22 ]…”

Section: De Novo Rna Synthesis At Dsbs: An Unexpected Discoverymentioning

confidence: 99%

“…The micro RNA (miRNA) pathway is governed by two main RNase III enzymes, Drosha and Dicer, that cleave dsRNA substrates. It has been shown that a wide variety of Drosha and Dicer deficient cells display an impaired DDR response, [ 20,28,32–35 ] although the mechanisms behind this phenomenon are not well understood. Even though miRNAs do regulate cell cycle checkpoints and the abundance of some DDR effectors (reviewed in ref.…”

Section: Processing and Degradation Of Dilncrnasmentioning

confidence: 99%

RNA at DNA Double‐Strand Breaks: The Challenge of Dealing with DNA:RNA Hybrids

2020

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RNA polymerase II is recruited to DNA double-strand breaks (DSBs), transcribes the sequences that flank the break and produces a novel RNA type that has been termed damage-induced long non-coding RNA (dilncRNA). DilncRNAs can be processed into short, miRNA-like molecules or degraded by different ribonucleases. They can also form double-stranded RNAs or DNA:RNA hybrids. The DNA:RNA hybrids formed at DSBs contribute to the recruitment of repair factors during the early steps of homologous recombination (HR) and, in this way, contribute to the accuracy of the DNA repair. However, if not resolved, the DNA:RNA hybrids are highly mutagenic and prevent the recruitment of later HR factors. Here recent discoveries about the synthesis, processing, and degradation of dilncRNAs are revised. The focus is on RNA clearance, a necessary step for the successful repair of DSBs and the aim is to reconcile contradictory findings on the effects of dilncRNAs and DNA:RNA hybrids in HR.

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Drosha drives the formation of DNA:RNA hybrids around DNA break sites to facilitate DNA repair

Cited by 186 publications

References 74 publications

Control of ribosomal protein synthesis by Microprocessor complex

Control of ribosomal protein synthesis by Microprocessor complex

Chromatin-associated MRN complex protects highly transcribing genes from genomic instability

RNA at DNA Double‐Strand Breaks: The Challenge of Dealing with DNA:RNA Hybrids

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