While alkylated mRNAs are known to activate ribosome quality control in the cytoplasm, how do cells deal with damaged RNAs in the nucleus? In their current work, Tsao et al. discover a new pathway of RNA damage repair and unexpectedly find that RNA alkylation can induce R-loops and DNA breaks.

In all domains of life, the ancient K homology (KH) domain superfamily is central to RNA processes including splicing, transcription, posttranscriptional gene regulation, signaling, and translation. Proteins with 1 to 15 KH domains bind single-strand (ss) RNA or DNA with base sequence specificity. Here, we examine over 40 KH domain experimental structures in complex with nucleic acid (NA) and define a novel Helix-clasp-Helix-Strand-Loop (HcH-SL) NA recognition motif binding 4 to 5 nucleotides using 10 to 18 residues. HcH-SL includes and extends the Gly-X-X-Gly (GXXG) signature sequence "clasp" that brings together two helices as an ∼90° helical corner. The first helix primarily provides side chain interactions to unstack and sculpt 2 to 3 bases on the 5' end for recognition of sequence and chemistry. The clasp and second helix amino dipole recognize a central phosphodiester. Following the helical corner, a beta strand and its loop extension recognize the two 3' nucleotides, primarily through main chain interactions. The HcH-SL structural motif forms a right-handed triangle and concave functional interface for NA interaction that unexpectedly splays four bound nucleotides into conformations matching RNA recognition motif (RRM) bound RNA structures. Evolutionary analyses and its ability to recognize base sequence and chemistry make HcH-SL a primordial NA binding motif distinguished by its binding mode from other NA structural recognition motifs: helix-turn-helix, helix-hairpin-helix, and beta strand RRM motifs. Combined results explain its vulnerability as a viral hijacking target and how mutations and expression defects lead to diverse diseases spanning cancer, cardiovascular, fragile X syndrome, neurodevelopmental disorders, and paraneoplastic disease.

Genomic instability poses a formidable threat to cellular vitality and wellbeing, prompting cells to deploy an intricate DNA damage response (DDR) mechanism. Recent evidence has suggested that RNA is intricately linked to the DDR by serving as template, scaffold, or regulator during the repair of DNA damage. Additionally, RNA molecules undergo modifications, contributing to the epitranscriptome, a dynamic regulatory layer influencing cellular responses to genotoxic stress. The intricate interplay between RNA and the DDR sheds new light on how the RNA epigenome contributes to the maintenance of genomic integrity and ultimately shapes the fate of damaged cells.

N1-methyladenosine (m1A) is a conserved modification on house-keeping RNAs, including tRNAs and rRNAs. With recent advancement on m1A detection and mapping, m1A is revealed to have a secret life with regulatory functions. This includes the regulation of its canonical substrate tRNAs, and expands into new territories such as tRNA fragments, mRNAs and repeat RNAs. The dynamic regulation of m1A has been shown in different biological contexts, including stress response, diet, T cell activation and aging. Interestingly, m1A can also be installed by non-enzymatic mechanisms. However, technical challenges remain in m1A site mapping; as a result, controversies have been observed across different labs or different methods. In this review we will summarize the recent development of m1A detection, its dynamic regulation, and its biological functions on diverse RNA substrates.

Certain environmental toxins and chemotherapeutics are nucleic acid-damaging agents, causing adducts in DNA and RNA. While most of these adducts occur in RNA, the consequences of RNA damage are largely unexplored. Here, we demonstrate that nuclear RNA damage can result in loss of genome integrity in human cells. Specifically, we show that YTHDC1 regulates alkylation damage responses with the THO complex (THOC). In addition to its established binding to N6-methyladenosine (m6A), YTHDC1 binds to chemically induced N1-methyladenosine (m1A). Without YTHDC1, cells have greater alkylation damage sensitivity and increased DNA breaks, which are rescued by an RNA-specific dealkylase. These RNA-damage-induced DNA breaks (RDIBs) depend on R-loop formation, which is converted to DNA breaks by the XPG nuclease. Strikingly, in the absence of YTHDC1 or THOC, a nuclear RNA m1A methyltransferase is sufficient to induce DNA breaks. Our results provide mechanistic insight into how damaged RNAs can impact genomic integrity.

Endogenous and environmental stressors can damage DNA and RNA to compromise genome and transcriptome stability and integrity in cells, leading to genetic instability and diseases. Recent studies have demonstrated that RNA damage can also modulate genome stability via RNA-templated DNA synthesis, suggesting that it is essential to maintain RNA integrity for the sustainment of genome stability. However, little is known about RNA damage and repair and their roles in modulating genome stability. Current efforts have mainly focused on revealing RNA surveillance pathways that detect and degrade damaged RNA, while the critical role of RNA repair is often overlooked. Due to their abundance and susceptibility to nucleobase damaging agents, it is essential for cells to evolve robust RNA repair mechanisms that can remove RNA damage, maintaining RNA integrity during gene transcription. This is supported by the discovery of the alkylated RNA nucleobase repair enzyme human AlkB homolog 3 that can directly remove the methyl group on damaged RNA nucleobases, predominantly in the nucleus of human cells, thereby restoring the integrity of the damaged RNA nucleobases. This is further supported by the fact that several DNA repair enzymes can also process RNA damage. In this review, we discuss RNA damage and its effects on cellular function, DNA repair, genome instability, and potential RNA damage repair mechanisms. Our review underscores the necessity for future research on RNA damage and repair and their essential roles in modulating genome stability.

Mutation or deletion of the deubiquitinase USP7 causes Hao-Fountain syndrome (HAFOUS), which is characterized by speech delay, intellectual disability, and aggressive behavior and highlights important unknown roles of USP7 in the nervous system. Here, we conditionally delete USP7 in glutamatergic neurons in the mouse forebrain, triggering disease-relevant phenotypes, including sensorimotor deficits, impaired cognition, and aggressive behavior. Although USP7 deletion induces p53-dependent neuronal apoptosis, most behavioral abnormalities in USP7 conditional knockout mice persist following p53 loss. Strikingly, USP7 deletion perturbs the synaptic proteome and dendritic spinogenesis independent of p53. Integrated proteomics and biochemical analyses identify the RNA splicing factor Ppil4 as a key substrate of USP7. Ppil4 knockdown phenocopies the effect of USP7 loss on dendritic spines. Accordingly, USP7 loss disrupts splicing of synaptic genes. These findings reveal that USP7-Ppil4 signaling regulates neuronal connectivity in the developing brain with implications for our understanding of HAFOUS pathogenesis and other neurodevelopmental disorders.

RNA plays a central role in protein biosynthesis and performs diverse regulatory and catalytic functions, making it essential for all processes of life. Like DNA, RNA is constantly subjected to damage from endogenous and environmental sources. However, while the DNA damage response has been extensively studied, it was long assumed that RNA lesions are relatively inconsequential due to the transient nature of most RNA molecules. Here, we review recent studies that challenge this view by revealing complex RNA damage responses that determine survival when cells are exposed to nucleic acid-damaging agents and promote the resolution of RNA lesions.

All cells are exposed to chemicals that can damage their nucleic acids. Cells must protect these polymers because they code for key factors or complexes essential for life. Much of the work on nucleic acid damage has naturally focused on DNA, partly due to the connection between mutagenesis and human disease, especially cancer. Recent work has shed light on the importance of RNA damage, which triggers a host of conserved RNA quality control mechanisms. Because many RNA species are transient, and because of their ability to be retranscribed, RNA damage has largely been ignored. Yet, because of the connection between damaged RNA and DNA during transcription, and the association between essential complexes that process or decode RNAs, notably spliceosomes and ribosomes, the appropriate handling of damaged RNAs is critical for maintaining cellular homeostasis. This notion is bolstered by disease states, including neurodevelopmental and neurodegenerative diseases, that may arise upon loss or misregulation of RNA quality control mechanisms.

Complex pathways involving the DNA damage response (DDR) contend with cell-intrinsic and -extrinsic sources of DNA damage. DDR mis-regulation results in genome instability that can contribute to aging and diseases including cancer and neurodegeneration. Recent studies have highlighted key roles for several RNA species in the DDR, including short RNAs and RNA/DNA hybrids (R-loops) at DNA break sites, all contributing to efficient DNA repair. RNAs can undergo more than 170 distinct chemical modifications. These RNA modifications have emerged as key orchestrators of the DDR. Here, we highlight the function of enzyme- and non-enzyme-induced RNA modifications in the DDR, with particular emphasis on m6A, m5C, and RNA editing. We also discuss stress-induced RNA damage, including RNA alkylation/oxidation, RNA-protein crosslinks, and UV-induced RNA damage. Uncovering molecular mechanisms that underpin the contribution of RNA modifications to DDR and genome stability will have direct application to disease and approaches for therapeutic intervention.

Compared to other types of lung cancer, small cell lung cancer (SCLC) exhibits aggressive characteristics that promote drug resistance. Despite platinum-etoposide chemotherapy combined with immunotherapy being the current standard treatment, the rapid development of drug resistance has led to unsatisfactory clinical outcomes. This review focuses on the mechanisms contributing to the chemotherapy resistance phenotype in SCLC, such as increased intra-tumoral heterogeneity, alterations in the tumor microenvironment, changes in cellular metabolism, and dysregulation of apoptotic pathways. A comprehensive understanding of these drug resistance mechanisms in SCLC is imperative for ushering in a new era in cancer research, which will promise revolutionary advancements in cancer diagnosis and treatment methodologies.

Precise control of protein ubiquitination is essential for brain development, and hence, disruption of ubiquitin signaling networks can lead to neurological disorders. Mutations of the deubiquitinase USP7 cause the Hao-Fountain syndrome (HAFOUS), characterized by developmental delay, intellectual disability, autism, and aggressive behavior. Here, we report that conditional deletion of USP7 in excitatory neurons in the mouse forebrain triggers diverse phenotypes including sensorimotor deficits, learning and memory impairment, and aggressive behavior, resembling clinical features of HAFOUS. USP7 deletion induces neuronal apoptosis in a manner dependent of the tumor suppressor p53. However, most behavioral abnormalities in USP7 conditional mice persist despite p53 loss. Strikingly, USP7 deletion in the brain perturbs the synaptic proteome and dendritic spine morphogenesis independently of p53. Integrated proteomics analysis reveals that the neuronal USP7 interactome is enriched for proteins implicated in neurodevelopmental disorders and specifically identifies the RNA splicing factor Ppil4 as a novel neuronal substrate of USP7. Knockdown of Ppil4 in cortical neurons impairs dendritic spine morphogenesis, phenocopying the effect of USP7 loss on dendritic spines. These findings reveal a novel USP7-Ppil4 ubiquitin signaling link that regulates neuronal connectivity in the developing brain, with implications for our understanding of the pathogenesis of HAFOUS and other neurodevelopmental disorders.

The ring finger protein 113A (RNF113A) serves as an E3 ubiquitin ligase and a subunit of the spliceosome. Mutations in the RNF113A gene are associated with X-linked trichothiodystrophy (TTD). However, the cellular roles of RNF113A remain largely unknown. In this study, we performed transcriptome profiling of RNF113A knockout (KO) HeLa cells using RNA sequencing and revealed the upregulation of NRF2 pathway-associated genes. Further analysis confirmed that the KO of RNF113A promotes nuclear localization of the NRF2 protein and elevates the mRNA levels of NRF2 target genes. RNF113A KO cells showed high levels of intracellular reactive oxygen species (ROS) and decreased resistance to cell death following H2O2 treatment. Additionally, RNF113A KO cells more sensitively formed stress granules (SGs) under arsenite-induced oxidative stress. Moreover, RNF113A KO cells exhibited a decrease in glutathione levels, which could be attributed to a reduction in GLUT1 expression levels, leading to decreased glucose uptake reactions and lower intracellular glucose levels. These alterations potentially caused a reduction in ROS scavenging activity. Taken together, our findings suggest that the loss of RNF113A promotes oxidative stress-mediated activation of the NRF2 pathway, providing novel insights into RNF113A-associated human diseases.

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.

N1 -methyladenosine (m1 A) is a prevalent post-transcriptional RNA modification, and the distribution and dynamics of the modification play key epitranscriptomic roles in cell development. At present, the human AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family member ALKBH3 is the only known mRNA m1 A demethylase, but its catalytic mechanism remains unclear. Here, we present the structures of ALKBH3-oligo crosslinked complexes obtained with the assistance of a synthetic antibody crystallization chaperone. Structural and biochemical results showed that ALKBH3 utilized two β-hairpins (β4-loop-β5 and β'-loop-β'') and the α2 helix to facilitate single-stranded substrate binding. Moreover, a bubble-like region around Asp194 and a key residue inside the active pocket (Thr133) enabled specific recognition and demethylation of m1 A- and 3-methylcytidine (m3 C)-modified substrates. Mutation of Thr133 to the corresponding residue in the AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family members FTO or ALKBH5 converted ALKBH3 substrate selectivity from m1 A to N6 -methyladenosine (m6 A), as did Asp194 deletion. Our findings provide a molecular basis for understanding the mechanisms of substrate recognition and m1 A demethylation by ALKBH3. This study is expected to aid structure-guided design of chemical probes for further functional studies and therapeutic applications.

The central dogma of molecular biology posits that genetic information flows unidirectionally, from DNA, to RNA, and finally to protein. However, this directionality is broken in some cases, such as reverse transcription where RNA is converted to DNA by retroviruses and certain transposable elements. Our genomes have evolved and adapted to the presence of reverse transcription. Similarly, our genome is continuously maintained by several repair pathways to reverse damage due to various endogenous and exogenous sources. More recently, evidence has revealed that RNA, while in certain contexts may be detrimental for genome stability, is involved in promoting certain types of DNA repair. Depending on the pathway in question, the size of these DNA repair-associated RNAs range from one or a few ribonucleotides to long fragments of RNA. Moreover, RNA is highly modified, and RNA modifications have been revealed to be functionally associated with specific DNA repair pathways. In this review, we highlight aspects of this unexpected layer of genomic maintenance, demonstrating how RNA may influence DNA integrity.

The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC). TTDN1 loss, or a mutated IDR, causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. A Ttdn1-deficient mouse model recapitulates intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Fusing DBR1 to the TTDN1 IDR is sufficient to recruit DBR1 to the IBC and circumvents the functional requirement for TTDN1. Collectively, our findings link RNA lariat processing with splicing outcomes by revealing the molecular function of TTDN1.

Activating signal co-integrator 1 complex (ASCC) subunit 3 (ASCC3) supports diverse genome maintenance and gene expression processes, and contains tandem Ski2-like NTPase/helicase cassettes crucial for these functions. Presently, the molecular mechanisms underlying ASCC3 helicase activity and regulation remain unresolved. We present cryogenic electron microscopy, DNA-protein cross-linking/mass spectrometry as well as in vitro and cellular functional analyses of the ASCC3-TRIP4 sub-module of ASCC. Unlike the related spliceosomal SNRNP200 RNA helicase, ASCC3 can thread substrates through both helicase cassettes. TRIP4 docks on ASCC3 via a zinc finger domain and stimulates the helicase by positioning an ASC-1 homology domain next to the C-terminal helicase cassette of ASCC3, likely supporting substrate engagement and assisting the DNA exit. TRIP4 binds ASCC3 mutually exclusively with the DNA/RNA dealkylase, ALKBH3, directing ASCC3 for specific processes. Our findings define ASCC3-TRIP4 as a tunable motor module of ASCC that encompasses two cooperating NTPase/helicase units functionally expanded by TRIP4.

The primary method for probing DNA replication dynamics is DNA fiber analysis, which utilizes thymidine analog incorporation into nascent DNA, followed by immunofluorescent microscopy of DNA fibers. Besides being time-consuming and prone to experimenter bias, it is not suitable for studying DNA replication dynamics in mitochondria or bacteria, nor is it adaptable for higher-throughput analysis. Here, we present mass spectrometry-based analysis of nascent DNA (MS-BAND) as a rapid, unbiased, quantitative alternative to DNA fiber analysis. In this method, incorporation of thymidine analogs is quantified from DNA using triple quadrupole tandem mass spectrometry. MS-BAND accurately detects DNA replication alterations in both the nucleus and mitochondria of human cells, as well as bacteria. The high-throughput capability of MS-BAND captured replication alterations in an E. coli DNA damage-inducing gene library. Therefore, MS-BAND may serve as an alternative to the DNA fiber technique, with potential for high-throughput analysis of replication dynamics in diverse model systems.

The dynamic chemical modifications of DNA, RNA, and proteins can transform normal cells into malignant ones. While the DNA and protein modifications in cancer have been described extensively in the literature, there are fewer reports about the role of RNA modifications in cancer. There are over 100 forms of RNA modifications and one of these, mRNA methylation, plays a critical role in the malignant properties of the cells. mRNA methylation is a reversible modification responsible for regulating protein expression at the post-transcriptional level. Despite being discovered in the 1970s, a complete understanding of the different proteins involved and the mechanism behind mRNA methylation remains largely unknown. However, these mRNA methylations have been shown to foster cancer hallmarks via specific cellular targets inside the cell. In this review, we provide a brief overview of mRNA methylation and its emerging role in regulating the various hallmarks of cancer.

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.

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.

Cellular RNAs are modified by both physiological factors and exogenous agents, such as methyl methanesulfonate (MMS). However, techniques for analyzing how proteins may interact with these modified RNAs are limited. Here, we provide a protocol combining RNA immunoprecipitation (RIP) with mass spectrometry (MS) to analyze the methylation state of the RNAs bound by Flag-tagged proteins in mammalian cells. The approach is highly quantitative and can simultaneously detect several methylated nucleosides in a single experiment. For complete details on the use and execution of this protocol, please refer to Tsao et al. (2021).

N1-methyladenosine (m1A) is a prevalent and reversible post-transcriptional RNA modification that decorates tRNA, rRNA and mRNA. Recent studies based on technical advances in analytical chemistry and high-throughput sequencing methods have revealed the crucial roles of m1A RNA modification in gene regulation and biological processes. In this review, we focus on progress in the study of m1A methyltransferases, m1A demethylases and m1A-dependent RNA-binding proteins and highlight the biological mechanisms and functions of m1A RNA modification, as well as its association with human disease. We also summarize the current understanding of detection approaches for m1A RNA modification.

The activation of molecular oxygen for the highly selective functionalization and repair of DNA and RNA nucleobases is achieved by α-ketoglutarate (α-KG)/iron-dependent dioxygenases. Enzymes of special interest are the human homologs AlkBH of Escherichia coli EcAlkB and ten-eleven translocation (TET) enzymes. These enzymes are involved in demethylation or dealkylation of DNA and RNA, although additional physiological functions are continuously being revealed. Given their importance, studying enzyme-substrate interactions, turnover and kinetic parameters is pivotal for the understanding of the mode of action of these enzymes. Diverse analytical methods, including X-ray crystallography, UV/Vis absorption, electron paramagnetic resonance (EPR), circular dichroism (CD) and NMR spectroscopy have been employed to study the changes in the active site and the overall enzyme structure upon substrate, cofactor and inhibitor addition. Several methods are now available to assess activity of these enzymes. By discussing limitations and possibilities of these techniques for EcAlkB, AlkBH and TET we aim to give a comprehensive synopsis from a bioinorganic point of view, addressing researchers from different disciplines working in the highly interdisciplinary and rapidly evolving field of epigenetic processes and DNA/RNA repair and modification.