m⁶A RNA methylation regulates the fate of endogenous retroviruses

Abstract

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...

Exon-intron architecture determines mRNA stability by dictating m6A deposition

“…m6A, which - similar to m5C - is installed by default and imposes silencing but at the RNA level, may serve as a post-transcriptional counterpart. This idea resonates with recent literature implicating m6A in the silencing of transposable elements (Chelmicki et al, 2020; Chen et al, 2021; Liu et al, 2020a, 2021; Xu et al, 2021).…”

Potential role of m6A RNA methylation regulators in osteosarcoma and its clinical prognostic value

“…M6A modification on mRNA, similar to DNA and protein modification, can be reversibly and dynamically regulated by methyltransferases (writers), m6A-binding proteins (readers), and demethylases (erasers) [ 6 ]. Methyltransferase is an important class of catalytic enzymes, which can cause the m6A methylation in mRNA, mainly including METTL3, METTL14, METTL16, WTAP, VIRMA, RBM15, and ZC3H13 [ 7 – 10 ]. Readers are specific RNA binding proteins required by the mRNA modified by m6A to perform specific biological functions, including IGF2BPs, YTHDFs, YTHDCs, FMR1, and HNRNPs [ 11 – 13 ].…”

Endogenous retroviruses in the origins and treatment of cancer

“…Here we will briefly focus on the ERV control mechanisms that presently show the most potential as targets for genetic or pharmacological therapies (Table 2 ). Programmed mammalian ERV repression is enacted through the interconnected PIWI-interacting RNA (piRNA), DNA (CpG) methylation, chromatin packaging, and KAP1 (TRIM28) pathways, amongst others [ 76 , 111 ]. piRNAs are predominantly found in male germ cells and function to silence TEs, in part, by recruiting DNA methylation [ 112 , 113 ].…”