A common transcriptional mechanism involving R-loop and RNA abasic site regulates an enhancer RNA of APOE.

RNA is modified by hundreds of chemical reactions and folds into innumerable shapes. However, the regulatory role of RNA sequence and structure and how dysregulation leads to diseases remain largely unknown. Here, we uncovered a mechanism where RNA abasic sites in R-loops regulate transcription by pausing RNA polymerase II. We found an enhancer RNA, AANCR, that regulates the transcription and expression of apolipoprotein E (APOE). In some human cells such as fibroblasts, AANCR is folded into an R-loop and modified by N-glycosidic cleavage; in this form, AANCR is a partially transcribed nonfunctional enhancer and APOE is not expressed. In contrast, in other cell types including hepatocytes and under stress, AANCR does not form a stable R-loop as its sequence is not modified, so it is transcribed into a full-length enhancer that promotes APOE expression. DNA sequence variants in AANCR are associated significantly with APOE expression and Alzheimer's Disease, thus AANCR is a modifier of Alzheimer's Disease. Besides AANCR, thousands of noncoding RNAs are regulated by abasic sites in R-loops. Together our data reveal the essentiality of the folding and modification of RNA in cellular regulation and demonstrate that dysregulation underlies common complex diseases such as Alzheimer's disease.

RNA abasic sites in yeast and human cells.

RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group. Using primary amine-based reagents that react with the aldehyde group, we uncovered evidence for abasic sites in nascent RNA, messenger RNA, and ribosomal RNA from yeast and human cells. Mass spectroscopic analysis confirmed the presence of RNA abasic sites. The RNA abasic sites were found to be coupled to R-loops. We show that human methylpurine DNA glycosylase cleaves N-glycosidic bonds on RNA and that human apurinic/apyrimidinic endonuclease 1 incises RNA abasic sites in RNA-DNA hybrids. Our results reveal that, in yeast and human cells, there are RNA abasic sites, and we identify a glycosylase that generates these sites and an AP endonuclease that processes them.

High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase.

Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.

Aicardi-Goutières syndrome gene Rnaseh2c is a metastasis susceptibility gene in breast cancer.

Breast cancer is the second leading cause of cancer-related deaths in the United States, with the majority of these deaths due to metastatic lesions rather than the primary tumor. Thus, a better understanding of the etiology of metastatic disease is crucial for improving survival. Using a haplotype mapping strategy in mouse and shRNA-mediated gene knockdown, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. We found that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity, and immunophenotyping and RNA-sequencing analysis revealed engagement of the T cell-mediated adaptive immune response. Furthermore, the cGAS-Sting pathway was not activated in the metastatic cancer cells used in this study, suggesting that the mechanism of immune response in breast cancer is different from the mechanism proposed for Aicardi-Goutières Syndrome, a rare interferonopathy caused by RNase H2 mutation. These results suggest an important novel, non-enzymatic role for RNASEH2C during breast cancer progression and add Rnaseh2c to a panel of genes we have identified that together could determine patients with high risk for metastasis. These results also highlight a potential new target for combination with immunotherapies and may contribute to a better understanding of the etiology of Aicardi-Goutières Syndrome autoimmunity.

The Balancing Act of Ribonucleotides in DNA.

The abundance of ribonucleotides in DNA remained undetected until recently because they are efficiently removed by the ribonucleotide excision repair (RER) pathway, a process similar to Okazaki fragment (OF) processing after incision by Ribonuclease H2 (RNase H2). All DNA polymerases incorporate ribonucleotides during DNA synthesis. How many, when, and why they are incorporated has been the focus of intense work during recent years by many labs. In this review, we discuss recent advances in ribonucleotide incorporation by eukaryotic DNA polymerases that suggest an evolutionarily conserved role for ribonucleotides in DNA. We also review the data that indicate that removal of ribonucleotides has an important role in maintaining genome stability.

Unlike the Escherichia coli counterpart, archaeal RNase HII cannot process ribose monophosphate abasic sites and oxidized ribonucleotides embedded in DNA.

The presence of ribonucleoside monophosphates (rNMPs) in nuclear DNA decreases genome stability. To ensure survival despite rNMP insertions, cells have evolved a complex network of DNA repair mechanisms, in which the ribonucleotide excision repair pathway, initiated by type 2 RNase H (RNase HII/2), plays a major role. We recently demonstrated that eukaryotic RNase H2 cannot repair damage, that is, ribose monophosphate abasic (both apurinic or apyrimidinic) site (rAP) or oxidized rNMP embedded in DNA. Currently, it remains unclear why RNase H2 is unable to repair these modified nucleic acids having either only a sugar moiety or an oxidized base. Here, we compared the endoribonuclease specificity of the RNase HII enzymes from the archaeon Pyrococcus abyssi and the bacterium Escherichia coli, examining their ability to process damaged rNMPs embedded in DNA in vitro We found that E. coli RNase HII cleaves both rAP and oxidized rNMP sites. In contrast, like the eukaryotic RNase H2, P. abyssi RNase HII did not display any rAP or oxidized rNMP incision activities, even though it recognized them. Notably, the archaeal enzyme was also inactive on a mismatched rNMP, whereas the E. coli enzyme displayed a strong preference for the mispaired rNMP over the paired rNMP in DNA. On the basis of our biochemical findings and also structural modeling analyses of RNase HII/2 proteins from organisms belonging to all three domains of life, we propose that RNases HII/2's dual roles in ribonucleotide excision repair and RNA/DNA hydrolysis result in limited acceptance of modified rNMPs embedded in DNA.

RNase H2-initiated ribonucleotide excision repair.

Ribonucleotides are incorporated into DNA by the replicative DNA polymerases at frequencies of about 2 per kb, which makes them by far the most abundant form of potential DNA damage in the cell. Their removal is essential for restoring a stable intact chromosome. Here, we present a complete biochemical reconstitution of the ribonucleotide excision repair (RER) pathway with enzymes purified from Saccharomyces cerevisiae. RER is most efficient when the ribonucleotide is incised by RNase H2, and further excised by the flap endonuclease FEN1 with strand displacement synthesis carried out by DNA polymerase δ, the PCNA clamp, its loader RFC, and completed by DNA ligase I. We observed partial redundancy for several of the enzymes in this pathway. Exo1 substitutes for FEN1 and Pol ε for Pol δ with reasonable efficiency. However, RNase H1 fails to substitute for RNase H2 in the incision step of RER.

Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2.

Ribonucleoside 5'-monophosphates (rNMPs) are the most common non-standard nucleotides found in DNA of eukaryotic cells, with over 100 million rNMPs transiently incorporated in the mammalian genome per cell cycle. Human ribonuclease (RNase) H2 is the principal enzyme able to cleave rNMPs in DNA. Whether RNase H2 may process abasic or oxidized rNMPs incorporated in DNA is unknown. The base excision repair (BER) pathway is mainly responsible for repairing oxidized and abasic sites into DNA. Here we show that human RNase H2 is unable to process an abasic rNMP (rAP site) or a ribose 8oxoG (r8oxoG) site embedded in DNA. On the contrary, we found that recombinant purified human apurinic/apyrimidinic endonuclease-1 (APE1) and APE1 from human cell extracts efficiently process an rAP site in DNA and have weak endoribonuclease and 3'-exonuclease activities on r8oxoG substrate. Using biochemical assays, our results provide evidence of a human enzyme able to recognize and process abasic and oxidized ribonucleotides embedded in DNA.

Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria.

The genetic information in mammalian mitochondrial DNA is densely packed; there are no introns and only one sizeable noncoding, or control, region containing key cis-elements for its replication and expression. Many molecules of mitochondrial DNA bear a third strand of DNA, known as "7S DNA," which forms a displacement (D-) loop in the control region. Here we show that many other molecules contain RNA as a third strand. The RNA of these R-loops maps to the control region of the mitochondrial DNA and is complementary to 7S DNA. Ribonuclease H1 is essential for mitochondrial DNA replication; it degrades RNA hybridized to DNA, so the R-loop is a potential substrate. In cells with a pathological variant of ribonuclease H1 associated with mitochondrial disease, R-loops are of low abundance, and there is mitochondrial DNA aggregation. These findings implicate ribonuclease H1 and RNA in the physical segregation of mitochondrial DNA, perturbation of which represents a previously unidentified disease mechanism.

Crystal structures of RNase H2 in complex with nucleic acid reveal the mechanism of RNA-DNA junction recognition and cleavage.

Two classes of RNase H hydrolyze RNA of RNA/DNA hybrids. In contrast to RNase H1 that requires four ribonucleotides for cleavage, RNase H2 can nick duplex DNAs containing a single ribonucleotide, suggesting different in vivo substrates. We report here the crystal structures of a type 2 RNase H in complex with substrates containing a (5')RNA-DNA(3') junction. They revealed a unique mechanism of recognition and substrate-assisted cleavage. A conserved tyrosine residue distorts the nucleic acid at the junction, allowing the substrate to function in catalysis by participating in coordination of the active site metal ion. The biochemical and structural properties of RNase H2 explain the preference of the enzyme for junction substrates and establish the structural and mechanistic differences with RNase H1. Junction recognition is important for the removal of RNA embedded in DNA and may play an important role in DNA replication and repair.

Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication.

Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication.

RNase H2 roles in genome integrity revealed by unlinking its activities.

Ribonuclease H2 (RNase H2) protects genome integrity by its dual roles of resolving transcription-related R-loops and ribonucleotides incorporated in DNA during replication. To unlink these two functions, we generated a Saccharomyces cerevisiae RNase H2 mutant that can resolve R-loops but cannot cleave single ribonucleotides in DNA. This mutant definitively correlates the 2-5 bp deletions observed in rnh201Δ strains with single rNMPs in DNA. It also establishes a connection between R-loops and Sgs1-mediated replication reinitiation at stalled forks and identifies R-loops uniquely processed by RNase H2. In mouse, deletion of any of the genes coding for RNase H2 results in embryonic lethality, and in humans, RNase H2 hypomorphic mutations cause Aicardi-Goutières syndrome (AGS), a neuroinflammatory disorder. To determine the contribution of R-loops and rNMP in DNA to the defects observed in AGS, we characterized in yeast an AGS-related mutation, which is impaired in processing both substrates, but has sufficient R-loop degradation activity to complement the defects of rnh201Δ sgs1Δ strains. However, this AGS-related mutation accumulates 2-5 bp deletions at a very similar rate as the deletion strain.

The structural and biochemical characterization of human RNase H2 complex reveals the molecular basis for substrate recognition and Aicardi-Goutières syndrome defects.

RNase H2 cleaves RNA sequences that are part of RNA/DNA hybrids or that are incorporated into DNA, thus, preventing genomic instability and the accumulation of aberrant nucleic acid, which in humans induces Aicardi-Goutières syndrome, a severe autoimmune disorder. The 3.1 Å crystal structure of human RNase H2 presented here allowed us to map the positions of all 29 mutations found in Aicardi-Goutières syndrome patients, several of which were not visible in the previously reported mouse RNase H2. We propose the possible effects of these mutations on the protein stability and function. Bacterial and eukaryotic RNases H2 differ in composition and substrate specificity. Bacterial RNases H2 are monomeric proteins and homologs of the eukaryotic RNases H2 catalytic subunit, which in addition possesses two accessory proteins. The eukaryotic RNase H2 heterotrimeric complex recognizes RNA/DNA hybrids and (5')RNA-DNA(3')/DNA junction hybrids as substrates with similar efficiency, whereas bacterial RNases H2 are highly specialized in the recognition of the (5')RNA-DNA(3') junction and very poorly cleave RNA/DNA hybrids in the presence of Mg(2+) ions. Using the crystal structure of the Thermotoga maritima RNase H2-substrate complex, we modeled the human RNase H2-substrate complex and verified the model by mutational analysis. Our model indicates that the difference in substrate preference stems from the different position of the crucial tyrosine residue involved in substrate binding and recognition.

Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD.

Human RNase H1 contains an N-terminal domain known as dsRHbd for binding both dsRNA and RNA/DNA hybrid. We find that dsRHbd binds preferentially to RNA/DNA hybrids by over 25-fold and rename it as hybrid binding domain (HBD). The crystal structure of HBD complexed with a 12 bp RNA/DNA hybrid reveals that the RNA strand is recognized by a protein loop, which forms hydrogen bonds with the 2'-OH groups. The DNA interface is highly specific and contains polar residues that interact with the phosphate groups and an aromatic patch that appears selective for binding deoxyriboses. HBD is unique relative to non-sequence-specific dsDNA- and dsRNA-binding domains because it does not use positive dipoles of alpha-helices for nucleic acid binding. Characterization of full-length enzymes with defective HBDs indicates that this domain dramatically enhances both the specific activity and processivity of RNase H1. Similar activity enhancement by small substrate-binding domains linked to the catalytic domain likely occurs in other nucleic acid enzymes.

RNase H1, the Gold Standard for R-Loop Detection.

RNase H1 has become an essential tool to uncover the physiological and pathological roles of R-loops, three-stranded structures consisting of and RNA-DNA hybrid opposite to a single DNA strand (ssDNA). RNase H1 degrades the RNA portion of the R-loops returning the two DNA strands to double-stranded form (dsDNA). Overexpression of RNase H1 in different systems has helped to address the questions of where R-loops are located, their abundance, and mechanisms of formation, stability, and degradation. In this chapter we review multiple studies that used RNase H1 as an instrument to investigate R-loops multiple functions and their relevance in health and diseases.

Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex.

Eukaryotic RNase H2 is a heterotrimeric enzyme. Here, we show that the biochemical composition and stoichiometry of the human RNase H2 complex is consistent with the properties previously deduced from genetic studies. The catalytic subunit of eukaryotic RNase H2, RNASEH2A, is well conserved and similar to the monomeric prokaryotic RNase HII. In contrast, the RNASEH2B and RNASEH2C subunits from human and Saccharomyces cerevisiae share very little homology, although they both form soluble B/C complexes that may serve as a nucleation site for the addition of RNASEH2A to form an active RNase H2, or for interactions with other proteins to support different functions. The RNASEH2B subunit has a PIP-box and confers PCNA binding to human RNase H2. Unlike Escherichia coli RNase HII, eukaryotic RNase H2 acts processively and hydrolyzes a variety of RNA/DNA hybrids with similar efficiencies, suggesting multiple cellular substrates. Moreover, of five analyzed mutations in human RNASEH2B and RNASEH2C linked to Aicardi-Goutières Syndrome (AGS), only one, R69W in the RNASEH2C protein, exhibits a significant reduction in specific activity, revealing a role for the C subunit in enzymatic activity. Near-normal activity of four AGS-related mutant enzymes was unexpected in light of their predicted impairment causing the AGS phenotype.

R-Loop Analysis by Dot-Blot.

The three-stranded nucleic acid structure, R-loop, is increasingly recognized for its role in gene regulation. Initially, R-loops were thought to be the by-products of transcription; but recent findings of fewer R-loops in diseased cells made it clear that R-loops have functional roles in a variety of human cells. Next, it is critical to understand the roles of R-loops and how cells balance their abundance. A challenge in the field is the quantitation of R-loops since much of the work relies on the S9.6 monoclonal antibody whose specificity for RNA-DNA hybrids has been questioned. Here, we use dot-blots with the S9.6 antibody to quantify R-loops and show the sensitivity and specificity of this assay with RNase H, RNase T1, and RNase III that cleave RNA-DNA hybrids, single-stranded RNA, and double-stranded RNA, respectively. This method is highly reproducible, uses general laboratory equipment and reagents, and provides results within two days. This assay can be used in research and clinical settings to quantify R-loops and assess the effect of mutations in genes such as senataxin on R-loop abundance.

Val143 of human ribonuclease H2 is not critical for, but plays a role in determining catalytic activity and substrate specificity.

Ribonuclease H2 (RNase H2) exhibits both single ribonucleotide excision activity (activity A) and RNA strand degrading activity (activity B). Val143 of human RNase H2 is located at the active site and is conserved in eukaryotic RNase H2. In this study, we explored the role of Val143 in catalytic activity and substrate specificity. Nineteen single variants at amino acid position 143 were expressed in E. coli, and all variants except for V143C and V143M were purified from the cells. When the activity of the wild-type human RNase H2 (WT) was set as 100%, the relative activities A and B of the 17 variants were in the range of 0.05-130 and 0.02-42%, respectively. When the ratio of the relative activity A to the relative activity B of WT was set as 1, the ratios of the 17 variants were in the range of 0.2-5.7. This indicates that valine is optimal for balancing the two activities. The ratios for V143Y and V143W were relatively high (5.6 and 5.5, respectively), suggesting that the bulky residues like tyrosine and tryptophan at position 143 caused steric hindrance with the 2'-OH of the sugar moiety of the ribonucleotide at the 5' side of the scissile phosphodiester bond. The ratio for V143Q was relatively low (0.2). These results suggested that Val143 is not critical for, but plays a role in determining catalytic activity and substrate specificity.

RNase H2-RED carpets the path to eukaryotic RNase H2 functions.

Eukaryotic RNases H2 have dual functions in initiating the removal of ribonucleoside monophosphates (rNMPs) incorporated by DNA polymerases during DNA synthesis and in cleaving the RNA moiety of RNA/DNA hybrids formed during transcription and retrotransposition. The other major cellular RNase H, RNase H1, shares the hybrid processing activity, but not all substrates. After RNase H2 incision at the rNMPs in DNA the Ribonucleotide Excision Repair (RER) pathway completes the removal, restoring dsDNA. The development of the RNase H2-RED (Ribonucleotide Excision Defective) mutant enzyme, which can process RNA/DNA hybrids but is unable to cleave rNMPs embedded in DNA has unlinked the two activities and illuminated the roles of RNase H2 in cellular metabolism. Studies mostly in Saccharomyces cerevisiae, have shown both activities of RNase H2 are necessary to maintain genome integrity and that RNase H1 and H2 have overlapping as well as distinct RNA/DNA hybrid substrates. In mouse RNase H2-RED confirmed that rNMPs in DNA during embryogenesis induce lethality in a p53-dependent DNA damage response. In mammalian cell cultures, RNase H2-RED helped identifying DNA lesions produced by Top1 cleavage at rNMPs and led to determine that RNase H2 participates in the retrotransposition of LINE-1 elements. In this review, we summarize the studies and conclusions reached by utilization of RNase H2-RED enzyme in different model systems.

Characterization of six recombinant human RNase H2 bearing Aicardi-Goutiéres syndrome causing mutations.

Mammalian RNase H2 is a heterotrimeric enzyme consisting of one catalytic subunit (A) and two accessory subunits (B and C). RNase H2 is involved in the removal of a single ribonucleotide embedded in genomic DNA and removal of RNA of RNA/DNA hybrids. In humans, mutation of the RNase H2 gene causes a severe neuroinflammatory disorder Aicardi-Goutières syndrome (AGS). Here, we examined the activity and stability of six recombinant human RNase H2 variants bearing one AGS-causing mutation, A-G37S (Gly37 in the A subunit is replaced with Ser), A-N212I, A-R291H, B-A177T, B-V185G, or C-R69W. The activity of A-G37S was 0.3-1% of that of the wild-type RNase H2 (WT), while those of other five variants were 51-120%. In circular dichroism measurement, the melting temperatures of variants were 50-53°C, lower than that of WT (56°C). These results suggested that A-G37S had decreased activity and stability than WT, while other five variants had decreased stability but retained activity. In gel filtration chromatography of the purified enzyme preparation, WT migrated as a heterotrimer, while A-R291H eluted in two separate peaks containing either the heterotrimer or only the A subunit, suggesting that some AGS-causing mutations affect the heterotrimer-forming stability of RNase H2.