Blerta Xhemalce

R loop homeostasis is critical for DNA double-strand break (DSB) repair; however, how R loops are resolved in this context is poorly understood. Here, we define HELZ as a unique RNA-DNA helicase that resolves R loops to facilitate homologous recombination (HR) repair. From a synthetic lethal etoposide resistance siRNA screen, we found that HELZ depletion causes R loop-mediated hypersensitivity to DSB-inducing agents, and HELZ localizes and binds to DSBs. HELZ preferentially binds to and unwinds RNA-DNA hybrids with 5’ssRNA overhangs to promote R loop resolution genome-wide and at DSBs. Interestingly, HELZ facilitates BRCA1 recruitment to DSBs by preventing R loop accumulation, thereby promoting DNA end resection and HR to prevent R loop mediated genomic instability. In summary, we show that HELZ resolves R loops critical for HR, thereby promoting genome stability and resistance to DSB-inducing agents.

Retrons can produce multicopy single-stranded DNA in cells through self-primed reverse transcription. However, their potential for inserting genetic cargos in eukaryotes remains largely unexplored. Here we report the discovery and engineering of highly efficient retron-based gene editors for mammalian cells and vertebrates. Through bioinformatic analysis of metagenomic data and functional screening, we identify retron reverse transcriptases that are highly active in mammalian cells. Rational design further improves the editing efficiency to levels comparable with conventional single-stranded oligodeoxynucleotide donors but from a genetically encoded cassette. Retron editors exhibit robust activity with Cas12a nuclease and Cas9 nickase, expanding the genomic target scope and bypassing the need for a DNA double-stranded break. Using a rationally engineered retron editor, we incorporate a split GFP epitope tag for live-cell imaging. Lastly, we develop an all-RNA delivery strategy to enable DNA-free gene editing in cells and vertebrate embryos. This work establishes retron editors as a versatile and efficient tool for precise gene editing.

N ⁴ -acetylcytidine (ac ⁴ C) is deposited on diverse RNAs by N -acetyltransferase 10 (NAT10), a protein with high biological relevance for aging and cancer. We performed a comprehensive survey of ac ⁴ C using metabolic labeling, sodium cyanoborohydride chemical treatment coupled to next-generation sequencing (NGS), and ac ⁴ C antibody–based cell and molecular biology techniques. Our analysis shows that NAT10-dependent ac ⁴ C-acetylation is robust in rRNA and specific tRNAs but low/spurious in mRNA. It also revealed an inflammatory signature and mutagenesis at transcriptionally active sites in NAT10-KO cells. This finding led us to explore the role of NAT10 in R-loops, which were recently linked to APOBEC3B-mediated mutagenesis. Our analysis showed that R-loops are ac ⁴ C-acetylated in a NAT10-dependent manner. Furthermore, NAT10 restrains the levels of R-loops at a subset of differentially expressed genes in a catalytic activity–dependent manner. Together with cellular biology data showing ac ⁴ C-modified RNA in endosomal structures, we propose that increased levels of ac ⁴ C-unmodified RNAs, likely derived from R-loops, in endosomal structures induce inflammatory responses.

LSG1 is a conserved GTPase involved in ribosome assembly. It is required for the eviction of the nuclear export adapter NMD3 from the pre-60S subunit in the cytoplasm. In human cells, LSG1 has also been shown to interact with vesicle-associated membrane protein-associated proteins (VAPs) that are found primarily on the endoplasmic reticulum. VAPs interact with a large host of proteins which contain FFAT motifs (two phenylalanines (FF) in an acidic tract) and are involved in many cellular functions including membrane traffic and regulation of lipid transport. Here, we show that human LSG1 binds to VAPs via a noncanonical FFAT-like motif. Deletion of this motif specifically disrupts the localization of LSG1 to the ER, without perturbing LSG1-dependent recycling of NMD3 in cells or modulation of LSG1 GTPase activity in vitro.

5′-end modifications play key roles in determining RNA fates. Phospho-methylation is a noncanonical cap occurring on either 5′-PPP or 5′-P ends. We used ChemRAP, in which affinity purification of cellular proteins with chemically synthesized modified RNAs is coupled to quantitative proteomics, to identify 5′-Pme “readers”. We show that 5′-Pme is directly recognized by EPRS, the central subunit of the multisynthetase complex (MSC), through its linker domain, which has previously been involved in key noncanonical EPRS and MSC functions. We further determine that the 5′-Pme writer BCDIN3D regulates the binding of EPRS to specific mRNAs, either at coding regions rich in MSC codons, or around start codons. In the case of LRPPRC (leucine-rich pentatricopeptide repeat containing), a nuclear-encoded mitochondrial protein associated with the French Canadian Leigh syndrome, BCDIN3D deficiency abolishes binding of EPRS around its mRNA start codon, increases its translation but ultimately results in LRPPRC mislocalization. Overall, our results suggest that BCDIN3D may regulate the translation of specific mRNA via RNA-5′-Pme.

The histone demethylase KDM5A erases histone H3 lysine 4 methylation, which is involved in transcription and DNA damage responses (DDRs). While DDR functions of KDM5A have been identified, how KDM5A recognizes DNA lesion sites within chromatin is unknown. Here, we identify two factors that act upstream of KDM5A to promote its association with DNA damage sites. We have identified a noncanonical poly(ADP-ribose) (PAR)–binding region unique to KDM5A. Loss of the PAR-binding region or treatment with PAR polymerase (PARP) inhibitors (PARPi’s) blocks KDM5A–PAR interactions and DNA repair functions of KDM5A. The histone variant macroH2A1.2 is also specifically required for KDM5A recruitment and function at DNA damage sites, including homology-directed repair of DNA double-strand breaks and repression of transcription at DNA breaks. Overall, this work reveals the importance of PAR binding and macroH2A1.2 in KDM5A recognition of DNA lesion sites that drive transcriptional and repair activities at DNA breaks within chromatin that are essential for maintaining genome integrity.

Long gone are the days when RNA was thought of as just an intermediate between DNA and proteins in the central dogma of molecular biology (DNA → RNA → Proteins). RNA has never ceased to surprise scientists throughout the past seven decades of molecular biology, from the postulation and discovery of transfer RNA in the 1950s to the emergence of RNA therapeutics jump-started by the COVID-19 global pandemic in 2020. One of most exiting surprises of the last decade was the realization that RNA modifications play key roles in regulating the fates of RNAs through the same rules of engagement as other epigenetic mechanisms, i.e. through writers, erasers and readers of RNA modifications, as initially proposed by Chuan He in his grand challenge commentary entitled ‘RNA epigenetics?’ in Nature Chemical Biology in 2010 [ 1]. The first papers showing RNA modification patterns of m⁶A on mammalian messenger RNA at the transcriptome-wide level [ 2, 3], allowed the re-discovery of seminal papers from the 1970s to 2000s, as well as the birth of the new field of epitranscriptomic research (reviewed in [ 4]). The discovery of the roles of phospho-methylation on microRNA processing [ 5], or of m⁵C on tRNA fragment generation [ 6] and vault RNA processing [ 7] further suggested that all classes of RNAs may be controlled by a variety of RNA modifications. After almost a decade from these initial papers, we have compiled a collection of easy-to-read reviews focusing on key RNA modifications and their molecular and biological functions.