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Aberrant RNA methylation triggers recruitment of an alkylation repair complex

ABSTRACT: Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.

ORGANISM(S): Homo sapiens

PROVIDER: GSE158090 | GEO | 2021/02/01

REPOSITORIES: GEO

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Project description:Maintaining the integrity of nucleic acids is an essential feature of all organisms. Unwanted modification of DNA, if left unrepaired, is deleterious to cellular homeostasis and could have far-reaching consequences that include genomic instability and accumulation of mutations. Similarly, accumulation of damaged RNA has been correlated with various neurodegenerative diseases. Given their inherent reactivity, nucleic acids are susceptible to damage from both endogenous and exogenous reactive oxygen species (ROS) as well as alkylation agents. We have recently begun to address how some of these modifications on mRNA impact the function of the ribosome and in particular the decoding process. We find that most modifications severely change the speed and accuracy of translation. These observations revealed that oxidation of RNA is most likely to stall the ribosome and necessitates the presence of quality-control processes to handle damaged mRNA. To this end, we have uncovered a connection between the process of no-go decay (NGD), which degrades mRNAs that stall translation, and chemical insults. In the absence of key NGD factors in yeast, the levels of damaged mRNA significantly increase and cells are rendered sensitive to oxidizing and alkylation agents. Furthermore, these agents activate ribosome quality control (RQC) of nascent peptides. Deletion of the E3 ligase responsible for the ubiquitination of the nascent peptides resulted in the accumulation of protein aggregates in the presence of oxidizing and alkylating agents. These observations suggest that chemical damage stalls translation, activating NGD and RQC. Interestingly, the addition of nucleic-acids-damaging agents was also found to result in K63-linked ubiquitination of ribosomal proteins. This signaling-mode of ubiquitination precedes DNA-damage marks, indicating that cells respond to RNA damage due to stalled ribosomes before DNA damage. Collectively our data highlights the burden of chemically-damaged mRNA on cellular homeostasis and suggests that organisms evolved ribosome-based mRNA-surveillance processes to rapidly degrade it.

Project description:Humans are exposed to a wide range of exogenous and endogenous alkylating agents, including agents frequently used as cytostatic drugs in cancer treatment (Drablos, Feyzi et al. 2004). Alkylating agents are mutagenic and cytotoxic and the major body of research has focused on their effects upon DNA. Less is known about the cellular consequences of alkylation to RNA, proteins and lipids, although such damage is likely quantitatively dominating due to the mere abundance of these macromolecules. DNA and RNA can be alkylated by SN1 and SN2 alkylating agents and alkylation occurs at nucleophilic O and N-atoms in the nucleosides as well as at O atoms in the phosphodiester bonds (Shooter, Howse et al. 1974, Sedgwick 2004). While alkylation at the O atoms in the nucleobases (O6G, O4T and O2C) are not affected by their hydrogen bonding, N1A and N3C contain an electron pair involved in hydrogen bonding. Alkylation at these nitrogens are thus more frequent in RNA and single-stranded DNA than in double stranded DNA (Bodell and Singer 1979). To cope with genomic damage inflicted by alkylating agents, a set of complex signal transduction pathways and DNA repair enzymes, collectively called the DNA damage response (DDR) is essential. The DDR senses the DNA damage and starts a highly choreographed response in order to resolve DNA damage or -replication issues (Ciccia and Elledge 2010). Specific repair mechanisms also exist to repair alkylated RNA (Aas, Otterlei et al. 2003, Wurtmann and Wolin 2009), although the biological significance of such repair yet remains elusive. Nevertheless, alkylation damage has been shown to alter the base-pairing properties of RNA bases and can interfere with tRNA-rRNA (Yoshizawa, Fourmy et al. 1999) as well as mRNA-tRNA (Ougland, Zhang et al. 2004){Hudson, 2015 #259}. Likely, other RNA functions that relies on base pairing might also be affected by alkylation damage, e.g. the correct folding of tRNAs and the function of miRNAs. Alkylation of mRNA may also affect translation indirectly by affecting binding affinities to RNA binding proteins (RBPs), as recently demonstrated for 6-meA. This enzymatically induced modification promotes binding of the 6-maA reader proteins YTHDF1 and 2 . While YTHDF2 reduces the stability of 6-maA modified transcripts (Wang, Lu et al. 2014), YTHDF1 actively promotes protein synthesis by interacting with the translation machinery (Wang, Zhao et al. 2015). To what degree alkylation damage to mRNA may promote similar effects by modulating translation remains, however, to be investigated. Interestingly, several studies have identified RBPs as important players in the DDR (Beli, Lukashchuk et al. 2012, Jungmichel, Rosenthal et al. 2013). Here, RBPs appear to have three major roles: 1) regulate gene expression at both the transcriptional- and post-transcriptional level (Keene and Tenenbaum 2002, Glisovic, Bachorik et al. 2008, Rinn and Chang 2012), 2) prevent formation of R-loops (Skourti-Stathaki and Proudfoot 2014), and 3) participate directly in DNA repair (Montecucco and Biamonti 2013, Dutertre, Lambert et al. 2014, Naro, Bielli et al. 2015). Two large-scale mass spectrometry (MS) based studies of RBPs in human cancer cell lines together identified more than 1100 RBPs (Baltz, Munschauer et al. 2012, Castello, Fischer et al. 2012) and more recently the “RBPomes” of mouse embryonic stem cells (Kwon, Yi et al. 2013) and yeast (Mitchell, Jain et al. 2013) have been published. However, functional studies describing how these complex protein networks are affected during genotoxic cell stress are still largely missing. One previous study has monitored changes in the RBPome of mouse embryonic fibroblasts subsequent to treatment with the topoisomerase inhibitor etoposide {Boucas, 2015 #112}. To our knowledge, however, no such studies have attempted to monitor altered global protein:RNA binding subsequent to treatment with alkylating agents. This could be particularly interesting since base methylation constitutes an important functional modification of RNA. In the present study we aimed at investigating this in more detail by SILAC-based quantitation of proteins differentially binding to poly(A)-RNA in HeLa cells at different time points after treatment with the alkylating agent methyl methanesulfonate.

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Aberrant RNA methylation triggers recruitment of an alkylation repair complex

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