YTHDC1 cooperates with the THO complex to prevent RNA damage-induced DNA breaks.

Certain environmental toxins are nucleic acid damaging agents, as are many chemotherapeutics used for cancer therapy. These agents induce various adducts in DNA as well as RNA. Indeed, most of the nucleic acid adducts (>90%) formed due to these chemicals, such as alkylating agents, occur in RNA 1 . However, compared to the well-studied mechanisms for DNA alkylation repair, the biological consequences of RNA damage are largely unexplored. Here, we demonstrate that RNA damage can directly result in loss of genome integrity. Specifically, we show that a human YTH domain-containing protein, YTHDC1, regulates alkylation damage responses in association with the THO complex (THOC) 2 . In addition to its established binding to N 6-methyladenosine (m6A)-containing RNAs, YTHDC1 binds to N 1-methyladenosine (m1A)-containing RNAs upon alkylation. In the absence of YTHDC1, alkylation damage results in increased alkylation damage sensitivity and DNA breaks. Such phenotypes are fully attributable to RNA damage, since an RNA-specific dealkylase can rescue these phenotypes. These R NA d amage-induced DNA b reaks (RDIBs) depend on R-loop formation, which in turn are processed by factors involved in transcription-coupled nucleotide excision repair. Strikingly, in the absence of YTHDC1 or THOC, an RNA m1A methyltransferase targeted to the nucleus is sufficient to induce DNA breaks. Our results uncover a unique role for YTHDC1-THOC in base damage responses by preventing RDIBs, providing definitive evidence for how damaged RNAs can impact genomic integrity.

SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation-Phosphorylation Cross-talk.

Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of SMYD3 in cancer, uncovering this enzyme as a mediator of alkylation damage sensitivity and providing a rationale for small-molecule SMYD3 inhibition to improve responses to established chemotherapy. SIGNIFICANCE: SCLC rapidly becomes resistant to conventional chemotherapy, leaving patients with no alternative treatment options. Our data demonstrate that SMYD3 upregulation and RNF113A methylation in SCLC are key mechanisms that control the alkylation damage response. Notably, SMYD3 inhibition sensitizes cells to alkylating agents and promotes sustained SCLC response to chemotherapy. This article is highlighted in the In This Issue feature, p. 2007.

The ASCC2 CUE domain in the ALKBH3-ASCC DNA repair complex recognizes adjacent ubiquitins in K63-linked polyubiquitin.

Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and even cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3-Activating Signal Cointegrator 1 Complex Subunit (ASCC) repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains via its coupling of ubiquitin conjugation to ER degradation (CUE) domain. The basis for polyubiquitin-binding specificity was unclear, because CUE domains in other proteins typically bind a single ubiquitin and do not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. The ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, whereas the contacts with the proximal ubiquitin are unique to ASCC2. Residues in the N-terminal portion of the ASCC2 α1 helix contribute to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 α1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response. Our study reveals the versatility of CUE domains in ubiquitin recognition.

Aberrant RNA methylation triggers recruitment of an alkylation repair complex.

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.