National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2017 Annual Report of the Division of Intramural Research

Physiological, Biochemical, and Molecular-Genetic Events Governing the Recognition and Resolution of RNA/DNA Hybrids

Robert Crouch
  • Robert J. Crouch, PhD, Head, Section on Formation of RNA
  • Susana M. Cerritelli, PhD, Staff Scientist
  • Naushaba Hasin, PhD, Postdoctoral Fellow
  • Shashikala Mishra, PhD, Postdoctoral Fellow
  • Ryo Uehara, PhD, Postdoctoral Fellow
  • Kiran Sakhuja, MS, MSc, Biologist

Damaged DNA is a leading causes of many human diseases and disorders. We study the formation and resolution of RNA/DNA hybrids, which occur during DNA and RNA synthesis. Such hybrid molecules may lead to increased DNA damage but may also play critical roles in normal cellular processes. We are interested in how RNA/DNA hybrids are resolved and in the role that ribonucleases H (RNases H) play in their elimination. Two classes of RNases H, Class I and Class II, are present in most organisms.

Human patients with mutations in RNASEH1 exhibit a typical mitochondrial muscular phenotype (Reference 1). Our studies were the first to show that RNase H1 is essential for maintenance of mitochondrial DNA. Mice deleted for the Rnaseh1 gene arrest embryonic development at day 8.5 as a result of failure to amplify mitochondrial DNA (Reference 2). Aicardi-Goutières syndrome (AGS), a severe neurological disorder with symptoms appearing at or soon after birth, can be caused by defective human RNase H2 (Reference 3). We are examining mouse models of AGS to gain insight into the human disorder. To understand the mechanisms, functions, substrates, and basic molecular genetics of RNases H, we employ molecular-genetic and biochemical tools in yeast and mouse models.

Contrasts between Class I and Class II RNases H

Many of our investigations over the past few years focused on RNase H1. RNase H1 recognizes the 2′-OH of four consecutive ribonucleotides, while the DNA strand is distorted to fit into a pocket of the enzyme. Thus, the enzyme requires more than one ribonucleotide for cleavage of RNA in RNA/DNA hybrids. In both eukaryotes and prokaryotes, RNases H1 consist of a single polypeptide. In contrast, RNase H2 is a complex of three distinct polypeptides in eukaryotes but a single polypeptide in prokaryotes. The catalytic subunit of the hetero-trimeric RNase H2 of eukaryotes is similar in its primary amino-acid sequence to the prokaryotic enzyme. RNase H2 can recognize and cleave both RNA/DNA hybrids and a single ribonucleotide (Reference 4) or the transition from the ribonucleotide in the case of RNA–primed DNA synthesis (e.g., rrrrrDDDD in DNA—italics indicate transition from ribonucleotide to deoxyribonucleotide).

Several types of RNA/DNA hybrid structures are formed, which are processed differently. Simple RNA/DNA hybrids consist of one strand of RNA paired with one strand of DNA. The HIV–AIDS reverse transcriptase (RT) forms such hybrids when copying its genomic RNA into DNA. The RT also has an RNase H domain that is structurally and functionally similar to the class I cellular RNase H and is necessary for several steps of viral DNA synthesis. R-loop hybrids (three-stranded nucleic acid structures) have two separated DNA strands, with one hybridized to RNA while the other is in a single-stranded form. These structures sometimes form during transcription and can lead to chromosomal breakage. However, they are also part of the normal process of switching (recombination) from one form of immunoglobulin to another, resulting in different isoforms of antibodies. Another form of hybrid are single or multiple ribonucleotides incorporated into DNA during replication (Reference 4). The first two types of hybrid are substrates for class I and II RNases H. The third is uniquely recognized by type 2 RNases H.

Dual activities of RNase H2; Aicardi-Goutières syndrome

Eukaryotic RNases H2 recognize and resolve RNA hybridized or covalently attached to DNA—two chemically distinct structures—using the same catalytic mechanism for hydrolysis. RNase H2 mutations that reduce catalytic activity, or fail to properly interact with in vivo substrates, cause Aicardi-Goutières syndrome (AGS). Mutations in seven genes are known to cause AGS, with more than 50% of AGS patients having mutations in any of the three subunits of RNase H2. We previously expressed (in Escherichia coli) and purified human RNases H2 with mutations corresponding to several of those seen in AGS patients, one having significant loss of RNase H2 activity, RNASEH2A-G37S (G37S). Using the 3D structure of the human enzyme we had determined, we could locate all known mutations in RNase H2 that cause AGS. The wide distribution of the mutations suggests that modest changes in stability, interaction with other unknown proteins, and loss of catalysis can all cause AGS. A mutation near the catalytic center of G37S found in some AGS patients results in low RNase H2 activity for both embedded ribonucleotides in DNA and RNA/DNA hybrids (Reference 3). We are developing mouse models of AGS to clarify which defects are associated with each RNase H2 activity.

Mice bearing the G37S mutation in homozygous form are perinatal lethal, i.e., either dead at birth or die within a few hours of birth (Reference 3). Mutations in another gene, TREX1, also cause AGS, and it has been shown that homozygous knockout (KO) mice are viable but die after a few weeks owing to a cardiomyopathy that can be prevented by either blocking an innate or adaptive immune response. In contrast, the G37S–mutant perinatal lethality and the fact that RNase H2 KO mice die early in embryogenesis suggest a more severe defect than that seen in TREX1–KO mice. We attempted to rescue the perinatal phenotype by eliminating one part of the innate immune pathway or by completely inactivating the adaptive immune response. Viability of these mice is no different from that of the innate or adaptive competent mice. It is possible that there are additional defects in G37S mice that are directly related to viability, not innate immunity. However, the expression of several interferon-stimulated genes (ISGs) is elevated in mouse embryonic fibroblasts (MEFs) derived from G37S homozygous embryos, supporting a role for innate immunity the AGS phenotype. Damaged DNA that finds its way to the cytoplasm can be sensed by the cGAS protein producing the small molecule cGAMP, which interacts with the Sting protein, an important protein for the DNA–sensing innate immune pathway. Mice that are homozygous for G37S and deleted for the cGAS or Sting genes are mostly perinatal lethal but no longer exhibit increases in ISGs. Interestingly, a small fraction of the double G37S–Sting KO are viable, indicating only limited involvement of ISGs in perinatal lethality (Reference 3). Further studies are under way, which we expect will lead us to the cause of lethality.

To distinguish the defects that persistent RNA/DNA hybrids and single ribonucleotides joined to DNA caused in vivo, Hyongi Chon, a former postdoctoral fellow, rationally designed a modified RNase H2 to make an enzyme unable to cleave single ribonucleotides embedded in DNA but which retained RNA/DNA hydrolytic activity. The mutant enzyme, which we call RED (Ribonucleotide Excision Deficient) resolves RNA/DNA hybrids, which are substrates of both RNase H1 and RNase H2. Unlike the mouse and human RNases H2, RNase H2 activity is not required in the yeast Saccharomyces cerevisiae. Employing the ease of genetic mutation studies in in yeast, we demonstrated that yeast producing the RNase H2RED enzyme acted in vivo leaving embedded ribonucleotides (rNMPs) in DNA but was potent in removal of RNA in RNA/DNA hybrids.

Embryonic lethality of mice Rnaseh2b–KO stains has been attributed to accumulation of rNMPs in DNA, but lethality could be the result of loss of RNA/DNA hydrolysis or a combination of both rNMP and RNA/DNA hydrolysis defects. To distinguish among the possible causes of embryonic lethality, we generated a mouse that produces the RNase H2RED enzyme. Mouse embryo fibroblasts (MEFs) derived from Rnaseh2RED mice have the same high level of rNMPs as seen in Rnaseh2bKO MEFs. Interestingly, the Rnaseh2RED mice die around the same time as the Rnaseh2b-KO mice. Therefore, lethality of the Knockout and RED RNase H2 mouse strains led to embryonic death. Rnaseh2aG37S/RED embryos also arrest at approximately the same as Rnaseh2aRED/RED embryos because of better association of RNase H2RED than RNase H2G37S to substrate with embedded rNMPs. The result is important because some RNase H2-AGS patients have similar compound heterozygous mutations in which there may be a dominant mutated enzyme.

Mitochondrial DNA, class switch recombination, and RNase H1

During embryonic development, RNase H1 is required for progress beyond day E8.5 (References 1,2). A single transcript of the mouse Rnaseh1 gene is translated to make two nearly identical proteins, one localizing to the nucleus and the other to the mitochondrion. We previously showed that the Rnaseh1–deleted embryos fail to amplify mtDNA (mitochondrial DNA), causing developmental arrest. Nuclear DNA replicated normally in Rnaseh1–KO embryos. We are examining loss of the Rnaseh1 gene during B cell development to follow the process of RNase H1 depletion in a simpler system. Following conditional deletion of Rnaseh1 at an early stage of B cell development in mouse B cells, we found that resting, naive B cells are formed but that they are unable to become activated to carry out class switch recombination (CSR) to other isotypes (e.g., IgG), and that sera from these mice have a major deficit in antibodies. We are currently determining whether the loss of mitochondrial DNA is the explanation for the inability of the resting B cells to be completely activated, as well as what changes in mRNA levels differ between the conditional KO and WT Rnaseh1 genes.

CSR occurs when B cells are stimulated with cytokines for growth and isotype switching. R-loops have been known to be present at the sites to be recombined. Transcription at the two sites required for recombination forms R-loops. Activation-induced cytidine deaminase (AID) initiates breakage by deamination of cytidines followed by recombinational events. AID also creates somatic hypermutations adding to the repertoire of possible antibodies. We collaborated with Frederic Chedin using DNA RNA immuno-precipitation sequence (DRIP-seq) to detect R-loop formation in the absence of RNase H1. In resting cells there are few R-loops at the site for CSR. However, there is an increase after stimulation, indicating the start of CSR but, owing to the absence of mtDNA function, the process is not completed. Current evidence indicates that RNA of the R-loops is removed by the RNA exosome but not by RNase H. We found that expression of very high levels of the nuclear form of RNase H1 removes R-loops (Reference 5). Frequency of CSR is not increased but AID gains access to the transcribed DNA strand, creating more somatic hypermutations (SHM)—apparently more efficiently than the RNA exosome. These findings emphasize the importance of transcription to form R-loops as the most important limitation on frequency of CSR. Stated another way; without transcription-generated R-loops AID can neither generate SHM nor CSR. The findings also support a model in which removal of RNA from the R-loops leaves the two DNA strands as single strands, possibly because of the formation of stable DNA structures such as G4 quadruplexes, and they are relevant to stable R-loops present at many promoter and 3′-UTR portions of genes. We are interested in determining how the R-loops are protected from normal levels of RNase H1 and whether this also occurs in CSR, where there appears to be insufficient RNase H1.

Publications

  1. Akmana G, Desaia R, Bailey LJ, Yasukawab T, Rosa ID, Durigon R, Holmes JB, Moss CF, Mennuni M, Houlden H, Crouch RJ, Hanna MG, Pitceathly RDS, Spinazzola A, Holt IJ. Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria. Proc Natl Acad Sci USA 2016 113:E4276-E4285.
  2. Holmes JB, Akman G, Wood SR, Sakhuja K, Cerritelli SM, Moss C, Bowmaker MR, Jacobs HT, Crouch RJ, Holt IJ. Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication. Proc Natl Acad Sci USA 2015 112:9334-9339.
  3. Pokatayev V, Hasin N, Chon H, Cerritelli SM, Yan N, Crouch RJ. RNase H2 catalytic core Aicardi-Goutières syndrome-related mutant invokes cGAS-STING innate immune sensing pathway in mice. J Exp Med 2016 213:329-336.
  4. Cerritelli SM, Crouch RJ. The balancing act of ribonucleotides in DNA. Trends Biochem Sci 2016 41:434-445.
  5. Maul RW, Chon H, Sakhuja K, Cerritelli SM, Gugliotti LA, Gearhart PJ, Crouch RJ. R-Loop depletion by over-expressed RNase H1 in mouse B cells increases activation-induced deaminase access to the transcribed strand without altering frequency of isotype switching. J Mol Biol 2017 429:3255-3263.

Collaborators

  • Frederic Chedin, PhD, University of California, Davis, Davis, CA
  • Patricia J. Gearhart, PhD, Laboratory of Molecular Biology and Immunology, NIA, Baltimore, MD
  • Ian J. Holt, PhD, Biodonostia Institute, Donostia, San Sebastián, Spain
  • Herbert C. Morse, MD, Laboratory of Immunopathology, NIAID, Bethesda, MD
  • Francesca Storici, PhD, School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA
  • Nan Yan, PhD, University of Texas Southwestern Medical Center, Dallas, TX
  • Kiyoshi Yasukawa, PhD, Kyoto University, Kyoto, Japan

Contact

For more information, email crouch@helix.nih.gov or visit http://sfr.nichd.nih.gov.

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