Biology subjects

Hartono, S. R.

Publications and source records attributed to Hartono, S. R..

8 recordsLinked to original sources

bioRxiv2025

RNase H1 levels dramatically affect mitochondrial genome maintenance with little impact on nuclear R-loops in murine B cells

Overexpression of RNase H1, a ribonuclease that degrades RNA:DNA hybrids and R-loops, can suppress genome instability phenotypes in a range of maladaptive conditions. This has been interpreted to suggest that genotoxic co-transcriptional R-loops arise under these conditions and are resolved by RNase H1. Here, we manipulated RNase H1 levels using conditional knockout and overexpression models in primary murine B cells and mapped the resulting genomic R-loop landscapes. Rnaseh1 deletion resulted in a dramatic loss of mitochondrial replication and compromised B cell responses, consistent with a critical mitochondrial function for RNase H1. Genome-wide R-loops were, however, not significantly affected. More surprisingly, overexpressing active nuclear RNase H1 did not lead to significant reduction of R-loop levels or change their distribution. These results were confirmed using a human cell line in which active, nuclear RNase H1 can be induced. Our findings indicate that co-transcriptional R-loops are not efficiently resolved by RNase H1 and suggest that the identity of the RNA/DNA hybrids at the root of the genome instability phenotypes suppressed by RNase H1 may need to be re-interpreted.

genomics↗

bioRxiv2020

SF3B1-targeted Splicing Inhibition Triggers Global Alterations in Transcriptional Dynamics and R-Loop Metabolism

Efficient co-transcriptional splicing is thought to suppress genome-destabilizing R-loops. Inhibition of SF3B1, a core U2 spliceosome component, by Pladienolide B (PladB) in human K562 cells caused widespread intron retention and modest R-loops gains. Minimal overlap existed between these events, suggesting that unspliced introns by themselves do not cause excessive R-loops. R-loop gains were instead driven by extensive readthrough transcription at a subset of stress-response genes, defining a new class of aberrant "downstream of genes" (DoG) R-loops. Such DoG R-loops were temporally and spatially uncoupled from loci experiencing DNA damage. Unexpectedly, the predominant response to splicing inhibition was a global R-loop loss. This resulted from increased promoter-proximal pausing and defective transcription elongation associated with premature termination. Similar results were observed upon depletion of Aquarius, a U2 spliceosome-associated factor previously thought to suppress R-loops. Thus, U2 spliceosome-targeted splicing inhibition triggered profound alterations in transcriptional dynamics, leading to unexpected disruptions in R-loop landscapes. HIGHLIGHTSO_LIIntron retention caused by SF3B1 inhibition does not trigger excessive R-loops C_LIO_LIStress-response genes shows readthrough transcription and R-loop gains C_LIO_LIR-loop gains and DNA damage are temporally and spatially uncoupled C_LIO_LIU2 snRNP inhibition causes broad reduction in transcription and dominant R-loop loss C_LI

molecular biology↗

bioRxiv2020

NON-DENATURING BISULFITE TREATMENT AND SINGLE-MOLECULE REAL-TIME SEQUENCING REVEALS D-LOOP FOOTPRINTS, THEIR LENGTH, POSITION AND DISTRIBUTION

Displacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of a crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loop with base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint, revealing D-loop characteristics at unprecedented resolution. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

biochemistry↗

bioRxiv2020

Rdh54/Tid1 Inhibits Rad51-Rad54-Mediated D-loop Formation and Limits D-loop Length

Displacement loops (D-loops) are intermediates formed during homologous recombination that play a pivotal role in the fidelity of repair. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54 is also present in somatic cells where its function is less understood. While Rdh54 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54-mediated invasions. Here, we show that Rdh54 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54 uniquely restricts the lengths of Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast, whereas Rdh54 expression is suppressed in diploids. We propose that Rdh54 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54 acts as a physical roadblock to Rad54s translocation activity, limiting D-loop formation and D-loop length.

biochemistry↗

bioRxiv2020

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

The contribution of RNA:DNA hybrid metabolism to cellular processes and disease states has become a prominent topic of study. The S9.6 antibody recognizes RNA:DNA hybrids with a subnanomolar affinity, making it a broadly used tool to detect and study RNA:DNA hybrids. However, S9.6 also binds double-stranded RNA in vitro with significant affinity. Though frequently used in immunofluorescence microscopy, the possible reactivity of S9.6 with non-RNA:DNA hybrid substrates in situ, particularly RNA, has not been comprehensively addressed. Furthermore, S9.6 immunofluorescence microscopy has been methodologically variable and generated discordant imaging datasets. In this study, we find that the majority of the S9.6 immunofluorescence signal observed in fixed human cells arises from RNA, not RNA:DNA hybrids. S9.6 staining was quantitatively unchanged by pre-treatment with the human RNA:DNA hybrid-specific nuclease, RNase H1, despite experimental verification in situ that S9.6 could recognize RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to pre-treatments with RNase T1, and in some cases RNase III, two ribonucleases that specifically degrade single-stranded and double-stranded RNA, respectively. In contrast, genome-wide maps obtained by high-throughput DNA sequencing after S9.6-mediated DNA:RNA Immunoprecipitation (DRIP) are RNase H1-sensitive and RNase T1- and RNase III-insensitive. Altogether, these data demonstrate that the S9.6 antibody, though capable of recognizing RNA:DNA hybrids in situ and in vitro, suffers from a lack of specificity that precludes reliable imaging of RNA:DNA hybrids and renders associated imaging data inconclusive in the absence of controls for its promiscuous recognition of cellular RNAs.

molecular biology↗

bioRxiv2019

High-Throughput Single-Molecule R-loop Footprinting Reveals Principles of R-loop Formation

R-loops are a prevalent class of non-B DNA structures that form during transcription upon reannealing of the nascent RNA to the template DNA strand. R-loops have been profiled using the S9.6 antibody to immunoprecipitate DNA:RNA hybrids. S9.6-based DNA:RNA immunoprecipitation (DRIP) techniques revealed that R-loops form dynamically over conserved genic hotspots. We developed an orthogonal profiling methodology that queries R-loops via the presence of long stretches of single-stranded DNA on the looped-out strand. Non-denaturing sodium bisulfite treatment catalyzes the conversion of unpaired cytosines to uracils, creating permanent genetic tags for the position of an R-loop. Long read, single-molecule PacBio sequencing allows the identification of R-loop footprints at near nucleotide resolution in a strand-specific manner on single DNA molecules and at ultra-deep coverage. Single-molecule R-loop footprinting (SMRF-seq) revealed a strong agreement between S9.6-and bisulfite-based R-loop mapping and confirmed that R-loops form from unspliced transcripts over genic hotspots. Using the largest single-molecule R-loop dataset to date, we show that individual R-loops generate overlapping sets of molecular clusters that pile-up through larger R-loop-prone zones. SMRF-seq further established that R-loop distribution patterns are driven by both intrinsic DNA sequence features and DNA topological constraints, revealing the principles of R-loop formation.

genetics↗

bioRxiv2018

DNA Topoisomerase I differentially modulates R-loops across the human genome

BackgroundCo-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both positive and negative supercoils. How Top1 regulates R-loop structures at a global level is unknown.\n\nResultsHere, we performed high-resolution strand-specific R-loop mapping in human cells depleted for Top 1 and found that Top1 depletion resulted in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes. R-loop gains were characteristic for long, highly transcribed, genes located in gene-poor regions anchored to Lamin B1 domains and in proximity to H3K9me3-marked heterochromatic patches. R-loop losses, by contrast, occurred in gene-rich regions overlapping H3K27me3-marked active replication initiation regions. Interestingly, Top1 depletion coincided with a block of the cell cycle in G0/G1 phase and a trend towards replication delay.\n\nConclusionsOur findings reveal new properties of Top1 in regulating R-loop homeostasis and suggest a potential role for Top1 in controlling replication initiation via R-loop formation.

genomics↗

bioRxiv2017

The affinity of the S9.6 antibody for double-stranded RNAs impacts the mapping of R-loops in fission yeast.

R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions.

molecular biology↗