RNA-DNA Hybrid Research Articles

Resolving the polycistronic aftermath: Essential role of topoisomerase IA in preventing R-loops in Leishmania

Kinetoplastid parasites are ‘living bridges’ in the evolution from prokaryotes to higher eukaryotes. The near-intronless genome of the kinetoplastid Leishmania, exhibit polycistronic transcription which can facilitate R-loop formation. Therefore, to prevent such DNA-RNA hybrids Leishmania has retained prokaryotic-like DNA Topoisomerase IA (LdTOPIA) in course of evolution. LdTOPIA is an essential enzyme which is expressed ubiquitously and is adapted for the compartmentalized eukaryotic form in harboring functional bipartite nuclear localization signals. Although exhibiting greater homology to mycobacterial TOPIA, LdTOPIA could functionally complement the growth lethality of E. coli TOPIA null GyrB ts strain at non-permissive temperatures. Purified LdTOPIA exhibits, Mg2+ dependent relaxation of only negatively supercoiled DNA and preference towards single-stranded DNA substrates. LdTOPIA prevent nuclear R-loops as conditional LdTOPIA downregulated parasites exhibit R-loop formation and thereby parasite killing. The clinically used tricyclic antidepressant, norclomipramine could specifically inhibit LdTOPIA and lead to R-loop formation and parasite elimination. This comprehensive study therefore paves an avenue for drug repurposing against Leishmania.

R2D ligase: Unveiling a novel DNA ligase with surprising DNA-to-RNA ligation activity.

DNA ligases catalyze bond formation in the backbone of nucleic acids via the formation of a phosphodiester bond between adjacent 5' phosphates and 3' hydroxyl groups on one strand of the duplex. While DNA ligases preferentially ligate single breaks in double-stranded DNA (dsDNA), they are capable of ligating a multitude of other nucleic acid substrates like blunt-ended dsDNA, TA overhangs, short overhangs and various DNA-RNA hybrids. Here we report a novel DNA ligase from Cronobacter phage CR 9 (R2D Ligase) with an unexpected DNA-to-RNA ligation activity. The R2D ligase shows excellent efficiency when ligating DNA to either end of RNA molecules using a DNA template. Furthermore, we show that DNA can be ligated simultaneously to both the 5' and 3' ends of microRNA-like molecules in a single reaction mixture. Abortive adenylated side product formation is suppressed at lower ATP concentrations and the ligase reaction reaches near completion when ligating RNA-to-DNA or DNA-to-RNA. The ligation of a DNA strand to the 5'-PO4 2- end of RNA is unique among the commercially available ligases and may facilitate novel workflows in microRNA analysis, RNA sequencing and the preparation of chimeric guide DNA-RNA for gene editing applications.

Regulation of transcription patterns, poly(ADP-ribose), and RNA-DNA hybrids by the ATM protein kinase

The ataxia telangiectasia mutated (ATM) protein kinase is a master regulator of the DNA damage response and also an important sensor of oxidative stress. Analysis of gene expression in ataxia-telangiectasia (A-T) patient brain tissue shows that large-scale transcriptional changes occur in patient cerebellum that correlate with the expression level and guanine-cytosine (GC) content of transcribed genes. In human neuron-like cells in culture, we map locations of poly(ADP-ribose) and RNA-DNA hybrid accumulation genome-wide with ATM inhibition and find that these marks also coincide with high transcription levels, active transcription histone marks, and high GC content. Antioxidant treatment reverses the accumulation of R-loops in transcribed regions, consistent with the central role of reactive oxygen species in promoting these lesions. Based on these results, we postulate that transcription-associated lesions accumulate in ATM-deficient cells and that the single-strand breaks and PARylation at these sites ultimately generate changes in transcription that compromise cerebellum function and lead to neurodegeneration over time in A-T patients.

Gold Nanoparticle-Based Colorimetric and Fluorescent Dual-Mode Lateral Flow Immunoassay for SARS-CoV-2 Detection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection caused the COVID-19 pandemic, impacting the global economy and medical system due to its fast spread and extremely high infectivity. Efficient control of the spread of the disease relies on a fast, accurate, and convenient detection system for the early screening of the infected population. Although reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold-standard method for SARS-CoV-2 RNA analysis, it has complex experimental procedures and relies on expensive instruments and professional operators. In this work, we proposed a simple, direct, amplification-free lateral flow immunoassay (LFIA) with dual-mode detection of SARS-CoV-2 RNA via direct visualization as well as fluorescence detection. The viral RNA was detected by the designed DNA probes to specifically hybridize with the conserved open reading frame 1ab (ORF1ab), envelope protein (E), and nucleocapsid (N) regions of the SARS-CoV-2 genome to form DNA-RNA hybrids. These hybrids were then recognized by the dual-mode gold nanoparticles (DMNPs) to produce two different readout signals. The fluorescence characteristics of different sizes of GNPs were explored. Under the optimized conditions, the LFIA presented a linear detection range of 104-106 TU/mL with a limit of detection (LOD) of 0.76, 1.83, and 2.58 × 104 TU/mL for lentiviral particles carrying SARS-CoV-2 ORF1ab, E, and N motifs, respectively, in the fluorescent mode, which was up to 10 times more sensitive than the colorimetric mode. Furthermore, the LFIA exhibited excellent specificity to SARS-CoV-2 in comparison with other respiratory viruses. It could be used to detect SARS-CoV-2 in saliva samples. The developed LFIA represents a promising and convenient point-of-care method for dual-mode, rapid detection of SARS-CoV-2, especially in the periods with high infectivity.

Distinct negative elongation factor conformations regulate RNA polymerase II promoter-proximal pausing

Metazoan gene expression regulation involves pausing of RNA polymerase (Pol II) in the promoter-proximal region of genes and is stabilized by DSIF and NELF. Upon depletion of elongation factors, NELF appears to accompany elongating Pol II past pause sites; however, prior work indicates that NELF prevents Pol II elongation. Here, we report cryoelectron microscopy structures of Pol II-DSIF-NELF complexes with NELF in two distinct conformations corresponding to paused and poised states. The paused NELF state supports Pol II stalling, whereas the poised NELF state enables transcription elongation as it does not support a tilted RNA-DNA hybrid. Further, the poised NELF state can accommodate TFIIS binding to Pol II, allowing for Pol II reactivation at paused or backtracking sites. Finally, we observe that the NELF-A tentacle interacts with the RPB2 protrusion and is necessary for pausing. Our results define how NELF can support pausing, reactivation, and elongation by Pol II.

Interferon-γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein.

The ORF6 protein of the SARS-CoV-2 virus plays a crucial role in blocking the innate immune response of the infected cells by inhibiting interferon pathways. Additionally, it binds to and immobilises the RAE1 protein on the cytoplasmic membranes, thereby blocking mRNA transport from the nucleus to the cytoplasm. In all these cases, the host cell proteins are tethered by the flexible C-terminus of ORF6. A possible strategy to inhibit the biological activity of ORF6 is to bind its C-terminus with suitable ligands. Our in silico experiments suggest that hIFNγ binds the ORF6 protein with high affinity, thus impairing its interactions with RAE1 and, consequently, its activity in viral invasion. The in vitro studies reported here reveal a shift of the localisation of RAE1 in ORF6 overexpressing cells upon treatment with hIFNγ from predominantly cytoplasmic to mainly nuclear, resulting in the restoration of the export of mRNA from the nucleus. We also explored the expression of GFP in transfected-with-ORF6 cells by means of fluorescence microscopy and qRT-PCR, finding that treatment with hIFNγ unblocks the mRNA trafficking and reinstates the GFP expression level. The ability of the cytokine to block ORF6 is also reflected in minimising its negative effects on DNA replication by reducing accumulated RNA-DNA hybrids. Our results, therefore, suggest hIFNγ as a promising inhibitor of the most toxic SARS-CoV-2 protein.

Temperature-dependent elasticity of DNA, RNA, and hybrid double helices

Nucleic acid double helices in their DNA, RNA, and DNA-RNA hybrid form play a fundamental role in biology and are main building blocks of artificial nanostructures, but how their properties depend on temperature remains poorly understood. Here, we report thermal dependence of dynamic bending persistence length, twist rigidity, stretch modulus, and twist-stretch coupling for DNA, RNA, and hybrid duplexes between 7°C and 47°C. The results are based on all-atom molecular dynamics simulations using different force field parameterizations. We first demonstrate that unrestrained molecular dynamics can reproduce experimentally known mechanical properties of the duplexes at room temperature. Beyond experimentally known features, we also infer the twist rigidity and twist-stretch coupling of the hybrid duplex. As for the temperature dependence, we found that increasing temperature softens all the duplexes with respect to bending, twisting, and stretching. The relative decrease of the stretch moduli is 0.003–0.004/°C, similar for all the duplex variants despite their very different stretching stiffness, whereas RNA twist stiffness decreases by 0.003/°C, and smaller values are found for the other elastic moduli. The twist-stretch couplings are nearly unaffected by temperature. The stretching, bending, and twisting stiffness all include an important entropic component. Relation of our results to the two-state model of DNA flexibility is discussed. Our work provides temperature-dependent elasticity of nucleic acid duplexes at the microsecond scale relevant for initial stages of protein binding.

Super-resolution imaging and single-molecule tracking of RNA-DNA hybrid processing proteins in Bacillus subtilis

Super-resolution imaging and single-molecule tracking of RNA-DNA hybrid processing proteins in Bacillus subtilis

Rat1 promotes premature transcription termination at R-loops.

Certain DNA sequences can adopt a non-B form in the genome that interfere with DNA-templated processes, including transcription. Among the sequences that are intrinsically difficult to transcribe are those that tend to form R-loops, three-stranded nucleic acid structures formed by a DNA-RNA hybrid and the displaced ssDNA. Here we compared the transcription of an endogenous gene with and without an R-loop-forming sequence inserted. We show that, in agreement with previous in vivo and in vitro analyses, transcription elongation is delayed by R-loops in yeast. Importantly, we demonstrate that the Rat1 transcription terminator factor facilitates transcription throughout such structures by inducing premature termination of arrested RNAPIIs. We propose that RNase H degrades the RNA moiety of the hybrid, providing an entry site for Rat1. Thus, we have uncovered an unanticipated function of Rat1 as a transcription restoring factor opening up the possibility that it may also promote transcription through other genomic DNA structures intrinsically difficult to transcribe. If R-loop-mediated transcriptional stress is not relieved by Rat1, it will cause genomic instability, probably through the increase of transcription-replication conflicts, a deleterious situation that could lead to cancer.

DNA methylome, R-loop and clinical exome profiling of patients with sporadic amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the death of motor neurons, the aetiology of which is essentially unknown. Here, we present an integrative epigenomic study in blood samples from seven clinically characterised sporadic ALS patients to elucidate molecular factors associated with the disease. We used clinical exome sequencing (CES) to study DNA variants, DNA-RNA hybrid immunoprecipitation sequencing (DRIP-seq) to assess R-loop distribution, and reduced representation bisulfite sequencing (RRBS) to examine DNA methylation changes. The above datasets were combined to create a comprehensive repository of genetic and epigenetic changes associated with the ALS cases studied. This repository is well-suited to unveil new correlations within individual patients and across the entire patient cohort. The molecular attributes described here are expected to guide further mechanistic studies on ALS, shedding light on the underlying genetic causes and facilitating the development of new epigenetic therapies to combat this life-threatening disease.

Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts.

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.

The putative RNA helicase DDX1 associates with the nuclear RNA exosome and modulates RNA/DNA hybrids (R-loops)

The RNA exosome is a ribonuclease complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays roles in regulating gene expression and protecting the genome, including modulating the accumulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A) followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in double-strand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following double-strand breaks, and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing (DRIP-Seq). We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection.

Temperature-Dependent Twist of Double-Stranded RNA Probed by Magnetic Tweezer Experiments and Molecular Dynamics Simulations.

RNA plays critical roles in the transmission and regulation of genetic information and is increasingly used in biomedical and biotechnological applications. Functional RNAs contain extended double-stranded regions, and the structure of double-stranded RNA (dsRNA) has been revealed at high resolution. However, the dependence of the properties of the RNA double helix on environmental effects, notably temperature, is still poorly understood. Here, we use single-molecule magnetic tweezer measurements to determine the dependence of the dsRNA twist on temperature. We find that dsRNA unwinds with increasing temperature, even more than DNA, with ΔTwRNA = -14.4 ± 0.7°/(°C·kbp), compared to ΔTwDNA = -11.0 ± 1.2°/(°C·kbp). All-atom molecular dynamics (MD) simulations using a range of nucleic acid force fields, ion parameters, and water models correctly predict that dsRNA unwinds with rising temperature but significantly underestimate the magnitude of the effect. These MD data, together with additional MD simulations involving DNA and DNA-RNA hybrid duplexes, reveal a linear correlation between the twist temperature decrease and the helical rise, in line with DNA but at variance with RNA experimental data. We speculate that this discrepancy might be caused by some unknown bias in the RNA force fields tested or by as yet undiscovered transient alternative structures in the RNA duplex. Our results provide a baseline to model more complex RNA assemblies and to test and develop new parametrizations for RNA simulations. They may also inspire physical models of the temperature-dependent dsRNA structure.

Transcription as source of genetic heterogeneity in budding yeast.

Transcription presents challenges to genome stability both directly, by altering genome topology and exposing single-stranded DNA to chemical insults and nucleases, and indirectly by introducing obstacles to the DNA replication machinery. Such obstacles include the RNA polymerase holoenzyme itself, DNA-bound regulatory factors, G-quadruplexes and RNA-DNA hybrid structures known as R-loops. Here, we review the detrimental impacts of transcription on genome stability in budding yeast, as well as the mitigating effects of transcription-coupled nucleotide excision repair and of systems that maintain DNA replication fork processivity and integrity. Interactions between DNA replication and transcription have particular potential to induce mutation and structural variation, but we conclude that such interactions must have only minor effects on DNA replication by the replisome with little if any direct mutagenic outcome. However, transcription can significantly impair the fidelity of replication fork rescue mechanisms, particularly Break Induced Replication, which is used to restart collapsed replication forks when other means fail. This leads to de novo mutations, structural variation and extrachromosomal circular DNA formation that contribute to genetic heterogeneity, but only under particular conditions and in particular genetic contexts, ensuring that the bulk of the genome remains extremely stable despite the seemingly frequent interactions between transcription and DNA replication.

Efficient circular RNA synthesis through Gap-DNA splint-mediated ligation.

The COVID-19 pandemic heightened interest in circular RNA (C-RNA) for RNA therapeutics, offering advantages over linear mRNAs. Circular mRNA facilitates uncapped molecule development, and C-RNAs ensure stability in RNA interference therapeutics. The synthesis method, RNA ligation, is employed in C-RNA-based therapeutics. Stable DNA-RNA hybrid constructs enable efficient RNA ligase-based circularization.

Expression, purification, and biochemical analysis of the RNA-DNA hybrid helicase Sen1/SETX from Chaetomium thermophilum.

Yeast Sen1 and its vertebrate ortholog Senataxin (also known as SETX) are RNA-DNA resolving helicases. Sen1 and SETX are implicated in multiple critical nuclear functions not limited to but including DNA replication and repair, RNA processing, and transcription. These>200kDa helicases have a two-domain architecture with an N-terminal regulatory helical repeat array linked to an SF1b helicase motor core via a variable sized central linker of low complexity sequence. Given the size of these proteins, production of milligram quantities of protein that is suitable for biochemical, biophysical, and protein structural analysis has been challenging. To overcome these limitations, we developed a robust selectable high-yield YFP-fusion protein expression method for Sen1 production in mammalian cells, followed by purification on a high-affinity YFP-binding camelid nanobody support. Herein, we detail methods and protocols for the expression and purification of recombinant Sen1 from the thermophilic fungus Chaetomium thermophilum, and the quantitative characterization of its RNA-DNA duplex resolution activity.

Light-strand bias and enriched zones of embedded ribonucleotides are associated with DNA replication and transcription in the human-mitochondrial genome.

Abundant ribonucleoside-triphosphate (rNTP) incorporation into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA-structural change and genome instability. The rNMP distribution, characteristics, hotspotsand association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in hmtDNA of six different cell types. In most cell types, the rNMPs are preferentially embedded on the light strand of hmtDNA with a strong bias towards rCMPs; while in the liver-tissue cells, the rNMPs are predominately found on the heavy strand. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the control region, which links the rNMP presence to the frequent hmtDNA replication-failure events. We show a strong correlation between coding-sequence size and rNMP-embedment frequency per nucleotide on the non-template, light strand in all cell types, supporting the presence of transient RNA-DNA hybrids preceding light-strand replication. Moreover, we detect rNMP-embedment patterns that are only partly conserved across the different cell types and are distinct from those found in yeast mtDNA. The study opens new research directions to understand the biology of hmtDNA and genomic rNMPs.

S9.6-based hybrid capture immunoassay for pathogen detection

The detection of pathogens is critical for clinical diagnosis and public health surveillance. Detection is usually done with nucleic acid-based tests (NATs) and rapid antigen tests (e.g., lateral flow assays [LFAs]). Although NATs are more sensitive and specific, their use is often limited in resource-poor settings due to specialized requirements. To address this limitation, we developed a rapid DNA-RNA Hybrid Capture immunoassay (HC) that specifically detects RNA from pathogens. This assay utilizes a unique monoclonal antibody, S9.6, which binds DNA-RNA hybrids. Biotinylated single-stranded DNA probes are hybridized to target RNAs, followed by hybrid capture on streptavidin and detection with S9.6. The HC-ELISA assay can detect as few as 104 RNA molecules that are 2.2 kb in length. We also adapted this assay into a LFA format, where captured Bacillus anthracis rpoB RNA of 3.5 kb length was detectable from a bacterial load equivalent to 107 CFU per 100 mg of mouse tissue using either HC-ELISA or HC-LFA. Importantly, we also demonstrated the versatility of HC by detecting other pathogens, including SARS-CoV-2 and Toxoplasma gondii, showing its potential for broad pathogen detection. Notably, HC does not require amplification of the target nucleic acid and utilizes economical formats like ELISA and LFA, making it suitable for use in sentinel labs for pathogen detection or as a molecular tool in basic research laboratories. Our study highlights the potential of HC as a sensitive and versatile method for RNA-based pathogen detection.

Isolation and Enrichment of Extracellular Vesicles with Double-Positive Membrane Protein for Subsequent Biological Studies.

The isolation and enrichment of specific extracellular vesicle (EV) subpopulations are essential in the context of precision medicine. However, the current methods predominantly rely on a single-positive marker and are susceptible to interference from soluble proteins or impurities. This limitation represents a significant obstacle to the widespread application of EVs in biological research. Herein, a novel approach that utilizes proximity ligation assay (PLA) and DNA-RNA hybridization are proposed to facilitate the binding of two proteins on the EV membrane in advance enabling the isolation and enrichment of intact EVs with double-positive membrane proteins followed by using functionalized magnetic beads for capture and enzymatic cleavage for isolated EVs release. The isolated subpopulations of EVs can be further utilized for cellular uptake studies, high-throughput small RNA sequencing, and breast cancer diagnosis. Hence, developing and implementing a specialized system for isolating and enriching a specific subpopulation of EVs can enhance basic and clinical research in this field.

Long noncoding RNA-mediated epigenetic regulation of auxin-related genes controls shade avoidance syndrome in Arabidopsis.

The long noncoding RNA (lncRNA) AUXIN-REGULATED PROMOTER LOOP (APOLO) recognizes a subset of target loci across the Arabidopsis thaliana genome by forming RNA-DNA hybrids (R-loops) and modulating local three-dimensional chromatin conformation. Here, we show that APOLO regulates shade avoidance syndrome by dynamically modulating expression of key factors. In response to far-red (FR) light, expression of APOLO anti-correlates with that of its target BRANCHED1 (BRC1), a master regulator of shoot branching in Arabidopsis thaliana. APOLO deregulation results in BRC1 transcriptional repression and an increase in the number of branches. Accumulation of APOLO transcription fine-tunes the formation of a repressive chromatin loop encompassing the BRC1 promoter, which normally occurs only in leaves and in a late response to far-red light treatment in axillary buds. In addition, our data reveal that APOLO participates in leaf hyponasty, in agreement with its previously reported role in the control of auxin homeostasis through direct modulation of auxin synthesis gene YUCCA2, and auxin efflux genes PID and WAG2. We show that direct application of APOLO RNA to leaves results in a rapid increase in auxin signaling that is associated with changes in the plant response to far-red light. Collectively, our data support the view that lncRNAs coordinate shade avoidance syndrome in A. thaliana, and reveal their potential as exogenous bioactive molecules. Deploying exogenous RNAs that modulate plant-environment interactions may therefore become a new tool for sustainable agriculture.