Endogenous retroviruses (ERVs) are abundant and heterogenous groups of integrated retroviral sequences that impact genome regulation and cell physiology throughout their RNA-centered life cycle 1 . Failure to repress ERVs is associated with cancer, infertility, senescence and neurodegenerative diseases 2-4 . Here, using an unbiased genome-scale CRISPR knockout screen in mouse embryonic stem cells, we identify m 6 A RNA methylation as a novel means of ERV restriction. Methylation of ERV mRNAs is catalyzed by the complex of methyltransferase-like METTL3/METTL14 5 proteins whose depletion, along with their accessory subunits, WTAP and ZC3H13, led to increased mRNA abundance of Intracisternal A-particles (IAPs) and related ERVK elements specifically, by targeting their 5'UTR region. Using controlled auxindependent degradation of the METTL3/METTL14 enzymatic complex, we showed that IAP mRNA and protein abundance is dynamically and inversely correlated with m 6 A catalysis. By monitoring mRNA degradation rates upon METTL3/14 double degron, we further proved that m 6 A methylation destabilizes IAP transcripts. Finally, similarly to m 6 A writers, triple knockout of the m 6 A readers YTHDF1, DF2 and DF3 6 increased IAP mRNA abundance. This study sheds light onto a novel function of RNA methylation in protecting cellular integrity by clearing reactive ERV-derived RNA species, which may be especially important when transcriptional silencing is less stringent.

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...