A Class of Environmental and Endogenous Toxins Induces BRCA2 Haploinsufficiency and Genome Instability.

Mutations truncating a single copy of the tumor suppressor, BRCA2, cause cancer susceptibility. In cells bearing such heterozygous mutations, we find that a cellular metabolite and ubiquitous environmental toxin, formaldehyde, stalls and destabilizes DNA replication forks, engendering structural chromosomal aberrations. Formaldehyde selectively depletes BRCA2 via proteasomal degradation, a mechanism of toxicity that affects very few additional cellular proteins. Heterozygous BRCA2 truncations, by lowering pre-existing BRCA2 expression, sensitize to BRCA2 haploinsufficiency induced by transient exposure to natural concentrations of formaldehyde. Acetaldehyde, an alcohol catabolite detoxified by ALDH2, precipitates similar effects. Ribonuclease H1 ameliorates replication fork instability and chromosomal aberrations provoked by aldehyde-induced BRCA2 haploinsufficiency, suggesting that BRCA2 inactivation triggers spontaneous mutagenesis during DNA replication via aberrant RNA-DNA hybrids (R-loops). These findings suggest a model wherein carcinogenesis in BRCA2 mutation carriers can be incited by compounds found pervasively in the environment and generated endogenously in certain tissues with implications for public health.

Transient RNA-DNA Hybrids Are Required for Efficient Double-Strand Break Repair.

RNA-DNA hybrids are a major internal cause of DNA damage within cells, and their degradation by RNase H enzymes is important for maintaining genomic stability. Here, we identified an unexpected role for RNA-DNA hybrids and RNase H enzymes in DNA repair. Using a site-specific DNA double-strand break (DSB) system in Schizosaccharomyces pombe, we showed that RNA-DNA hybrids form as part of the homologous-recombination (HR)-mediated DSB repair process and that RNase H enzymes are essential for their degradation and efficient completion of DNA repair. Deleting RNase H stabilizes RNA-DNA hybrids around DSB sites and strongly impairs recruitment of the ssDNA-binding RPA complex. In contrast, overexpressing RNase H1 destabilizes these hybrids, leading to excessive strand resection and RPA recruitment and to severe loss of repeat regions around DSBs. Our study challenges the existing model of HR-mediated DSB repair and reveals a surprising role for RNA-DNA hybrids in maintaining genomic stability.

DICER ribonuclease removes harmful R-loops.

R-loops, which consist of a DNA-RNA hybrid and a displaced DNA strand, are known to threaten genome integrity. To counteract this, different mechanisms suppress R-loop accumulation by either preventing the hybridization of RNA with the DNA template (RNA biogenesis factors), unwinding the hybrid (DNA-RNA helicases), or degrading the RNA moiety of the R-loop (type H ribonucleases [RNases H]). Thus far, RNases H are the only nucleases known to cleave DNA-RNA hybrids. Now, we show that the RNase DICER also resolves R-loops. Biochemical analysis reveals that DICER acts by specifically cleaving the RNA within R-loops. Importantly, a DICER RNase mutant impaired in R-loop processing causes a strong accumulation of R-loops in cells. Our results thus not only reveal a function of DICER as an R-loop resolvase independent of DROSHA but also provide evidence for the role of multi-functional RNA processing factors in the maintenance of genome integrity in higher eukaryotes.

Structural basis for pegRNA-guided reverse transcription by a prime editor.

The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells1. However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9-M-MLV RTΔRNaseH-pegRNA-target DNA complex in multiple states. The termination structure, along with our functional analysis, reveals that M-MLV RT extends reverse transcription beyond the expected site, resulting in scaffold-derived incorporations that cause undesired edits at the target loci. Furthermore, structural comparisons among the pre-initiation, initiation and elongation states show that M-MLV RT remains in a consistent position relative to SpCas9 during reverse transcription, whereas the pegRNA-synthesized DNA heteroduplex builds up along the surface of SpCas9. On the basis of our structural insights, we rationally engineered pegRNA variants and prime-editor variants in which M-MLV RT is fused within SpCas9. Collectively, our findings provide structural insights into the stepwise mechanism of prime editing, and will pave the way for the development of a versatile prime editing toolbox.

Mind the Gapmer: Implications of Co-transcriptional Cleavage by Antisense Oligonucleotides.

While gapmers efficiently knock down as well as terminate transcription of nascent lncRNAs and mRNAs, Lee and Mendell (2020) and Lai et al. (2020) also demonstrate that Pol II termination is not observed with gapmers targeting the 3' terminal portions of the transcript.

Directed RNase H Cleavage of Nascent Transcripts Causes Transcription Termination.

An attractive approach to reduce gene expression is via the use of antisense oligonucleotides (ASOs) that harness the RNase H1 mechanism. Here we show that RNase H ASOs targeted to introns or exons robustly reduce the level of spliced RNA associated with chromatin. Surprisingly, intron-targeted ASOs reduce the level of pre-mRNA associated with chromatin to a greater extent than exon-targeted ASOs. This indicates that exon-targeted ASOs achieve full activity after the pre-mRNA has undergone splicing, but before the mRNA is released from chromatin. Even though RNase H ASOs can reduce the level of RNA associated with chromatin, the effect of ASO-directed RNA degradation on transcription has never been documented. Here we show that intron-targeted ASOs and, to a lesser extent, exon-targeted ASOs cause RNA polymerase II (Pol II) transcription termination in cultured cells and mice. Furthermore, ASO-directed transcription termination is mediated by the nuclear exonuclease XRN2.

METTL3 and N6-Methyladenosine Promote Homologous Recombination-Mediated Repair of DSBs by Modulating DNA-RNA Hybrid Accumulation.

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.

A unified allosteric/torpedo mechanism for transcriptional termination on human protein-coding genes.

The allosteric and torpedo models have been used for 30 yr to explain how transcription terminates on protein-coding genes. The former invokes termination via conformational changes in the transcription complex and the latter proposes that degradation of the downstream product of poly(A) signal (PAS) processing is important. Here, we describe a single mechanism incorporating features of both models. We show that termination is completely abolished by rapid elimination of CPSF73, which causes very extensive transcriptional readthrough genome-wide. This is because CPSF73 functions upstream of modifications to the elongation complex and provides an entry site for the XRN2 torpedo. Rapid depletion of XRN2 enriches these events that we show are underpinned by protein phosphatase 1 (PP1) activity, the inhibition of which extends readthrough in the absence of XRN2. Our results suggest a combined allosteric/torpedo mechanism, in which PP1-dependent slowing down of polymerases over termination regions facilitates their pursuit/capture by XRN2 following PAS processing.

RNase H2 degrades toxic RNA:DNA hybrids behind stalled forks to promote replication restart.

R-loops represent a major source of replication stress, but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest, and restart in Saccharomyces cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate their chromosomes normally under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Treated cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative intermediate in resuming replication. Similar impairments in nascent DNA resection and ssDNA formation at HU-arrested forks were observed in human cells lacking RNase H2. However, fork resection was fully restored by addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.

Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development.

The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.

Evolution of Inosine-Specific Endonuclease V from Bacterial DNase to Eukaryotic RNase.

Endonuclease V (EndoV) cleaves the second phosphodiester bond 3' to a deaminated adenosine (inosine). Although highly conserved, EndoV homologs change substrate preference from DNA in bacteria to RNA in eukaryotes. We have characterized EndoV from six different species and determined crystal structures of human EndoV and three EndoV homologs from bacteria to mouse in complex with inosine-containing DNA/RNA hybrid or double-stranded RNA (dsRNA). Inosine recognition is conserved, but changes in several connecting loops in eukaryotic EndoV confer recognition of 3 ribonucleotides upstream and 7 or 8 bp of dsRNA downstream of the cleavage site, and bacterial EndoV binds only 2 or 3 nt flanking the scissile phosphate. In addition to the two canonical metal ions in the active site, a third Mn2+ that coordinates the nucleophilic water appears necessary for product formation. Comparison of EndoV with its homologs RNase H1 and Argonaute reveals the principles by which these enzymes recognize RNA versus DNA.

R-Loops Promote Antisense Transcription across the Mammalian Genome.

Widespread antisense long noncoding RNA (lncRNA) overlap with many protein-coding genes in mammals and emanate from gene promoter, enhancer, and termination regions. However, their origin and biological purpose remain unclear. We show that these antisense lncRNA can be generated by R-loops that form when nascent transcript invades the DNA duplex behind elongating RNA polymerase II (Pol II). Biochemically, R-loops act as intrinsic Pol II promoters to induce de novo RNA synthesis. Furthermore, their removal across the human genome by RNase H1 overexpression causes the selective reduction of antisense transcription. Consequently, we predict that R-loops act to facilitate the synthesis of many gene proximal antisense lncRNA. Not only are R-loops widely associated with DNA damage and repair, but we now show that they have the capacity to promote de novo transcript synthesis that may have aided the evolution of gene regulation.

Selection pressures on evolution of ribonuclease H explored with rigorous free-energy-based design.

Understanding natural protein evolution and designing novel proteins are motivating interest in development of high-throughput methods to explore large sequence spaces. In this work, we demonstrate the application of multisite λ dynamics (MSλD), a rigorous free energy simulation method, and chemical denaturation experiments to quantify evolutionary selection pressure from sequence-stability relationships and to address questions of design. This study examines a mesophilic phylogenetic clade of ribonuclease H (RNase H), furthering its extensive characterization in earlier studies, focusing on E. coli RNase H (ecRNH) and a more stable consensus sequence (AncCcons) differing at 15 positions. The stabilities of 32,768 chimeras between these two sequences were computed using the MSλD framework. The most stable and least stable chimeras were predicted and tested along with several other sequences, revealing a designed chimera with approximately the same stability increase as AncCcons, but requiring only half the mutations. Comparing the computed stabilities with experiment for 12 sequences reveals a Pearson correlation of 0.86 and root mean squared error of 1.18 kcal/mol, an unprecedented level of accuracy well beyond less rigorous computational design methods. We then quantified selection pressure using a simple evolutionary model in which sequences are selected according to the Boltzmann factor of their stability. Selection temperatures from 110 to 168 K are estimated in three ways by comparing experimental and computational results to evolutionary models. These estimates indicate selection pressure is high, which has implications for evolutionary dynamics and for the accuracy required for design, and suggests accurate high-throughput computational methods like MSλD may enable more effective protein design.

A scalable and cost-efficient rRNA depletion approach to enrich RNAs for molecular biology investigations.

Transcriptomics analyses play pivotal roles in understanding the complex regulatory networks that govern cellular processes. The abundance of rRNAs, which account for 80%-90% of total RNA in eukaryotes, limits the detection and investigation of other transcripts. While mRNAs and long noncoding RNAs have poly(A) tails that are often used for positive selection, investigations of poly(A)- RNAs, such as circular RNAs, histone mRNAs, and small RNAs, typically require the removal of the abundant rRNAs for enrichment. Current approaches to deplete rRNAs for downstream molecular biology investigations are hampered by restrictive RNA input masses and high costs. To address these challenges, we developed rRNA Removal by RNaseH (rRRR), a method to efficiently deplete rRNAs from a wide range of human, mouse, and rat RNA inputs and of varying qualities at a cost 10- to 20-fold cheaper than other approaches. We used probe-based hybridization and enzymatic digestion to selectively target and remove rRNA molecules while preserving the integrity of non-rRNA transcripts. Comparison of rRRR to two commercially available approaches showed similar rRNA depletion efficiencies and comparable off-target effects. Our developed method provides researchers with a valuable tool for investigating gene expression and regulatory mechanisms across a wide range of biological systems at an affordable price that increases the accessibility for researchers to enter the field, ultimately advancing our understanding of cellular processes.

Profiling the interactome of oligonucleotide drugs by proximity biotinylation.

Drug-ID is a novel method applying proximity biotinylation to identify drug-protein interactions inside living cells. The covalent conjugation of a drug with a biotin ligase enables targeted biotinylation and identification of the drug-bound proteome. We established Drug-ID for two small-molecule drugs, JQ1 and SAHA, and applied it for RNaseH-recruiting antisense oligonucleotides (ASOs). Drug-ID profiles the drug-protein interactome de novo under native conditions, directly inside living cells and at pharmacologically effective drug concentrations. It requires minimal amounts of cell material and might even become applicable in vivo. We studied the dose-dependent aggregation of ASOs and the effect of different wing chemistries (locked nucleic acid, 2'-methoxyethyl and 2'-Fluoro) and ASO lengths on the interactome. Finally, we demonstrate the detection of stress-induced, intracellular interactome changes (actinomycin D treatment) with an in situ variant of the approach, which uses a recombinant biotin ligase and does not require genetic manipulation of the target cell.

RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops.

Telomeres-repeated, noncoding nucleotide motifs and associated proteins that are found at the ends of eukaryotic chromosomes-mediate genome stability and determine cellular lifespan1. Telomeric-repeat-containing RNA (TERRA) is a class of long noncoding RNAs (lncRNAs) that are transcribed from chromosome ends2,3; these RNAs in turn regulate telomeric chromatin structure and telomere maintenance through the telomere-extending enzyme telomerase4-6 and homology-directed DNA repair7,8. The mechanisms by which TERRA is recruited to chromosome ends remain poorly defined. Here we develop a reporter system with which to dissect the underlying mechanisms, and show that the UUAGGG repeats of TERRA are both necessary and sufficient to target TERRA to chromosome ends. TERRA preferentially associates with short telomeres through the formation of telomeric DNA-RNA hybrid (R-loop) structures that can form in trans. Telomere association and R-loop formation trigger telomere fragility and are promoted by the recombinase RAD51 and its interacting partner BRCA2, but counteracted by the RNA-surveillance factors RNaseH1 and TRF1. RAD51 physically interacts with TERRA and catalyses R-loop formation with TERRA in vitro, suggesting a direct involvement of this DNA recombinase in the recruitment of TERRA by strand invasion. Together, our findings reveal a RAD51-dependent pathway that governs TERRA-mediated R-loop formation after transcription, providing a mechanism for the recruitment of lncRNAs to new loci in trans.

Nucleolar RNA polymerase II drives ribosome biogenesis.

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.

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.

S-phase checkpoint prevents leading strand degradation from strand-associated nicks at stalled replication forks.

The S-phase checkpoint is involved in coupling DNA unwinding with nascent strand synthesis and is critical to maintain replication fork stability in conditions of replicative stress. However, its role in the specific regulation of leading and lagging strands at stalled forks is unclear. By conditionally depleting RNaseH2 and analyzing polymerase usage genome-wide, we examine the enzymology of DNA replication during a single S-phase in the presence of replicative stress and show that there is a differential regulation of lagging and leading strands. In checkpoint proficient cells, lagging strand replication is down-regulated through an Elg1-dependent mechanism. Nevertheless, when checkpoint function is impaired we observe a defect specifically at the leading strand, which was partially dependent on Exo1 activity. Further, our genome-wide mapping of DNA single-strand breaks reveals that strand discontinuities highly accumulate at the leading strand in HU-treated cells, whose dynamics are affected by checkpoint function and Exo1 activity. Our data reveal an unexpected role of Exo1 at the leading strand and support a model of fork stabilization through prevention of unrestrained Exo1-dependent resection of leading strand-associated nicks after fork stalling.

Multivalent DNAzyme agents for cleaving folded RNA.

Multivalent recognition and binding of biological molecules is a natural phenomenon that increases the binding stability (avidity) without decreasing the recognition specificity. In this study, we took advantage of this phenomenon to increase the efficiency and maintain high specificity of RNA cleavage by DNAzymes (Dz). We designed a series of DNA constructs containing two Dz agents, named here bivalent Dz devices (BDD). One BDD increased the cleavage efficiency of a folded RNA fragment up to 17-fold in comparison with the Dz of a conventional design. Such an increase was achieved due to both the improved RNA binding and the increased probability of RNA cleavage by the two catalytic cores. By moderating the degree of Dz agent association in BDD, we achieved excellent selectivity in differentiating single-base mismatched RNA, while maintaining relatively high cleavage rates. Furthermore, a trivalent Dz demonstrated an even greater efficiency than the BDD in cleaving folded RNA. The data suggests that the cooperative action of several RNA-cleaving units can significantly improve the efficiency and maintain high specificity of RNA cleavage, which is important for the development of Dz-based gene knockdown agents.