N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells

Abakir, Abdulkadir; Giles, Tom; Cristini, Agnese; Foster, Jeremy M.; Dai, Nan; Starczak, Marta; Rubio-Roldán, Alejandro; Li, Miaomiao; Eleftheriou, Maria; Crutchley, James; Flatt, Luke; Young, Lorraine; Gaffney, Daniel J; Denning, Chris; Dalhus, Bjørn; Emes, Richard D.; Gackowski, Daniel; Corrêa, Ivan R.; García‐Pérez, José L.; Klungland, Arne; Gromak, Natalia; Ruzov, Alexey

doi:10.1038/s41588-019-0549-x

Cited by 174 publications

(135 citation statements)

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“…3b and Extended Data Fig. 6a,b), we found that a significant number of METTL3-dependent m 6 A events mapped to retrotransposon annotations, comprising 13% of all peaks, including L1s—as previously reported 25,26 —and ERVK elements (Fig. 3b).…”

Section: Main Textsupporting

confidence: 82%

m⁶A RNA methylation regulates the fate of endogenous retroviruses

Chelmicki

Roger

Teissandier

et al. 2020

Preprint

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27 28 29 Endogenous retroviruses (ERVs) are abundant and heterogenous groups of integrated 30 retroviral sequences that impact genome regulation and cell physiology throughout 31 their RNA-centered life cycle 1 . Failure to repress ERVs is associated with cancer, 32 infertility, senescence and neurodegenerative diseases 2-4 . Here, using an unbiased 33 genome-scale CRISPR knockout screen in mouse embryonic stem cells, we identify 34 m 6 A RNA methylation as a novel means of ERV restriction. Methylation of ERV mRNAs 35 is catalyzed by the complex of methyltransferase-like METTL3/METTL14 5 proteins 36 whose depletion, along with their accessory subunits, WTAP and ZC3H13, led to 37 increased mRNA abundance of Intracisternal A-particles (IAPs) and related ERVK 38 elements specifically, by targeting their 5'UTR region. Using controlled auxin-39 dependent degradation of the METTL3/METTL14 enzymatic complex, we showed that 40 IAP mRNA and protein abundance is dynamically and inversely correlated with m 6 A 41 catalysis. By monitoring mRNA degradation rates upon METTL3/14 double degron, we 42 further proved that m 6 A methylation destabilizes IAP transcripts. Finally, similarly to 43 m 6 A writers, triple knockout of the m 6 A readers YTHDF1, DF2 and DF3 6 increased IAP 44 mRNA abundance. This study sheds light onto a novel function of RNA methylation in 45 protecting cellular integrity by clearing reactive ERV-derived RNA species, which may 46 be especially important when transcriptional silencing is less stringent.repressors, by transducing cells with single guide (sg)RNAs against the KRAB-associated 80 protein 1 (KAP1) 11 (Extended Data Fig. 1d,e, Supplementary Fig. 1, Supplementary Table 2, 81 Supplementary Table 3). 4For the screen, we transduced IAPEz-reporter cells with a lentiviral genome-wide 83 sgRNA library at multiplicity of infection (MOI)= 0.2-0.3 12 (Fig. 1b). Frequencies of sgRNAs 84 upon blasticidin selection (5, 7 and 9 days) versus non-selected conditions were assessed 85 via deep sequencing and candidate genes were identified using Model-based Analysis of 86 Genome-wide CRISPR/Cas9 Knockout (MAGeCK) 13 . Efficiency of selection was evidenced 87 by drop-out of control intergenic sgRNAs and genes were ranked based on sgRNA P-values 88 ( Fig. 1c, Supplementary Table 4, Supplementary Table 5). Although genome-wide screens of 89 this magnitude typically suffer from low-statistical confidence, we identified several-but not 90 all-genes previously associated with ERV repression (Resf1, Trp53, Daxx, Atrx, Uhrf1, 91 Cbx1, Dnmt1) 14-17 among the top 100 hits. Obtaining an incomplete list of known regulators 92 is a common outcome of genome-wide screens which can be due to multiple factors 93 including time-dependent dropout of essential genes with strong effects on cell viability (such 94 as Kap1) 18 , heterogeneity in sgRNA efficiencies or limited representation of sgRNAs. 95Nonetheless, we were able to identify several novel candidates for IAP control 96( Supplementary Table 4), offering a foundation for futu...

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Section: Main Textsupporting

confidence: 82%

m⁶A RNA methylation regulates the fate of endogenous retroviruses

Chelmicki

Roger

Teissandier

et al. 2020

Preprint

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“…While this work used normally cultured, unperturbed human cells, many studies have used S9.6-based imaging to measure changes in RNA:DNA hybrid content under various experimental conditions. An array of gene perturbations and drug treatments have been reported to cause changes in S9.6 IF signal attributed to altered R-loop metabolism or RNA:DNA hybrid levels [8,30,[34][35][36][47][48][49][50]. Many of the genes that have been ascribed roles in R-loop regulation participate in diverse RNA metabolic processes like transcription, RNA splicing, RNA processing, and RNA export [6].…”

Section: Discussionmentioning

confidence: 99%

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

Chédin

Smolka

Sanz

et al. 2020

Preprint

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The contribution of RNA:DNA hybrid metabolism to cellular processes and disease states has become a prominent topic of study. The S9.6 antibody recognizes RNA:DNA hybrids with a subnanomolar affinity, making it a broadly used tool to detect and study RNA:DNA hybrids. However, S9.6 also binds double-stranded RNA in vitro with significant affinity. Though frequently used in immunofluorescence microscopy, the possible reactivity of S9.6 with non-RNA:DNA hybrid substrates in situ, particularly RNA, has not been comprehensively addressed. Furthermore, S9.6 immunofluorescence microscopy has been methodologically variable and generated discordant imaging datasets. In this study, we find that the majority of the S9.6 immunofluorescence signal observed in fixed human cells arises from RNA, not RNA:DNA hybrids. S9.6 staining was quantitatively unchanged by pre-treatment with the human RNA:DNA hybrid-specific nuclease, RNase H1, despite experimental verification in situ that S9.6 could recognize RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to pre-treatments with RNase T1, and in some cases RNase III, two ribonucleases that specifically degrade singlestranded and double-stranded RNA, respectively. In contrast, genome-wide maps obtained by high-throughput DNA sequencing after S9.6-mediated DNA:RNA Immunoprecipitation (DRIP) are RNase H1-sensitive and RNase T1-and RNase IIIinsensitive. Altogether, these data demonstrate that the S9.6 antibody, though capable of recognizing RNA:DNA hybrids in situ and in vitro, suffers from a lack of specificity that precludes reliable imaging of RNA:DNA hybrids and renders associated imaging data inconclusive in the absence of controls for its promiscuous recognition of cellular RNAs.

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“…Furthermore, m 6 A has been shown to be present on RNA:DNA hybrids [ 136 , 137 ]. Abakir et al have shown that m 6 A can coordinate R-loop removal with the m 6 A reader, YTH-domain family member 2 (YTHDF2), contributing to genome stability [ 136 , 138 ]. Interestingly, this m 6 A modification of R-loops is not constitutive, but rather is cell cycle dependent.…”

Section: Looking Ahead: Rna Modifications and The Ddr A Role For Rnamentioning

confidence: 99%

“…Interestingly, this m 6 A modification of R-loops is not constitutive, but rather is cell cycle dependent. m 6 A is present on R-loops in S and G 2 /M phases of the cell cycle, and is reduced in the G 0 /G 1 phases of the cell cycle [ 136 , 138 ]. It is not yet clear if this could impact the DDR, in particular the DSB response, but given the known role of R-loops in DSB repair [ 63 , 100 , 111 ], it implicates a further possible role for m 6 A in the cellular DDR.…”

Section: Looking Ahead: Rna Modifications and The Ddr A Role For Rnamentioning

confidence: 99%

Jack of all trades? The versatility of RNA in DNA double-strand break repair

Ketley

Gullerová

2020

Essays in Biochemistry

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Abstract The mechanisms by which RNA acts in the DNA damage response (DDR), specifically in the repair of DNA double-strand breaks (DSBs), are emerging as multifaceted and complex. Different RNA species, including but not limited to; microRNA (miRNA), long non-coding RNA (lncRNA), RNA:DNA hybrid structures, the recently identified damage-induced lncRNA (dilncRNA), damage-responsive transcripts (DARTs), and DNA damage-dependent small RNAs (DDRNAs), have been shown to play integral roles in the DSB response. The diverse properties of these RNAs, such as sequence, structure, and binding partners, enable them to fulfil a variety of functions in different cellular contexts. Additionally, RNA can be modified post-transcriptionally, a process which is regulated in response to cellular stressors such as DNA damage. Many of these mechanisms are not yet understood and the literature contradictory, reflecting the complexity and expansive nature of the roles of RNA in the DDR. However, it is clear that RNA is pivotal in ensuring the maintenance of genome integrity. In this review, we will discuss and summarise recent evidence which highlights the roles of these various RNAs in preserving genomic integrity, with a particular focus on the emerging role of RNA in the DSB repair response.

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N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells

Cited by 174 publications

References 50 publications

m⁶A RNA methylation regulates the fate of endogenous retroviruses

m⁶A RNA methylation regulates the fate of endogenous retroviruses

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

Jack of all trades? The versatility of RNA in DNA double-strand break repair

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N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells

Cited by 174 publications

References 50 publications

m6A RNA methylation regulates the fate of endogenous retroviruses

m6A RNA methylation regulates the fate of endogenous retroviruses

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

Jack of all trades? The versatility of RNA in DNA double-strand break repair

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Product

Resources

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m⁶A RNA methylation regulates the fate of endogenous retroviruses

m⁶A RNA methylation regulates the fate of endogenous retroviruses