Explanation Research in the Section on Formation of RNA is directed toward understanding the interaction between RNA and DNA that perturb the DNA and how the cell responds to these RNA-containing DNAs. The AIDS virus, HIV, employs RNA as its genome and when copied into DNA RNA/DNA hybrids are intermediates that require RNase H activity, an enzyme that removes the RNA after it is copied by the viral DNA polymerase (Reverse Transcriptase or RT). RNase H is an essential part of RT and could be a target for therapeutic drugs. RNA/DNA hybrids are also present in normal cells, occasionally forming during transcription producing R-loops in which the RNA displaces one strand of DNA and forms a duplex with the complementary DNA strand. In addition, recombination proteins can form RNA/DNA hybrids by displacing one DNA strand while annealing complementary RNA to the other DNA strand. If unresolved, these R-loops lead to genome instability. The endogenous RNases H usually remove these R-loops. Aicardi Goutieres Syndrome (AGS) is a rare autoimmune disorder with severe neurological problems that can be caused by defects in human RNase H2. AGS mimics in utero viral infection including loss of white matter in the brain and producing high levels of interferon alpha in the cerebral spinal fluid. Mammalian RNases H2 can degrade RNA/DNA hybrids but can also recognize a single ribonucleotide in duplex DNA and initiates its removal. Current proposals suggest it is the failure to remove the missincorporated ribonucleotides results in DNA damage causing AGS. However, both RNA/DNA and single ribonucleotides in DNA remain when RNase H2 is not present;conditions tested so far. Thus, the jury is still out on the relationship between RNase H2 defects and AGS. We have been employed Saccharomyces cerevisiae as a model organism to examine conditions under which the two activities of RNase H2 are required. Interestingly, we have example where either RNase H1 or H2 can resolve the same R-loops and another in which R-loops are only degraded by RNase H2. To gain more insight into the effects of the same into AGS-related mutations in mammals, we have generated a mouse which expresses a mutant form of RNase H2 seen in a few AGS patients and have been examining the properties of the mouse and mouse tissues. The mice homozygous for the mutation are born dead or or die soon after birth. One model for defects in RNase H2 (and other proteins that when defective cause AGS) is activation of endogenous retroviruses which can result in induction of an innate immune response ultimately leading to an auto immune condition. While our results are still in an incomplete stage, we find no evidence of autoimmunity. RNase H1 is present in mitochondria (mt) and nuclei of mammalian cells and is required during embryonic development to generate mtDNA. Its function in the nucleus is not completely understood but it too is likely important for resolving R-loops with partial overlapping action with RNase H2. It has been shown that R-loop formation at repetitive sequences can lead to recombination that result in expansion (duplication) or deletion. Several human disorders arise when a triplet repeat such as CAG glutamine (Q) codon expand producing proteins containing long stretches of one amino acid (polyQ for CAG expansion). Often these proteins become aggregated and can affect the cell due to these complexes. In some cases, the expansion has direct affects on the function of the protein with the expansion. It is easy to imagine that the displaced DNA strand of R-loops can reanneal to its complementary DNA strand in multiple ways leading to repair and expansion or contraction. We are examining ataxic mice that arose in our RNase H1 knockout strain may have such triplet expansions. The role of RNase H1 in mtDNA replication is beginning to become clear from some of the studies carried out in collaboration with Ian Holt at the National Institute for Medical Research, MRC London. These advances have been made in part by using mouse embryo fibroblasts with defects in RNase H1 production that lead to cell death and accumulation of RNA/DNA in mtDNA. The model developed in Holts lab suggests RNA is a major intermediate in mtDNA replication, possibly by a mechanism similar to that described for RNA/DNA hybrid formation mediated by a recombination-related protein.

Project Start
Project End
Budget Start
Budget End
Support Year
42
Fiscal Year
2013
Total Cost
$1,210,070
Indirect Cost
City
State
Country
Zip Code
Gaidamakov, Sergei; Maximova, Olga A; Chon, Hyongi et al. (2014) Targeted deletion of the gene encoding the La autoantigen (Sjogren's syndrome antigen B) in B cells or the frontal brain causes extensive tissue loss. Mol Cell Biol 34:123-31
Cerritelli, Susana M; Crouch, Robert J (2009) Ribonuclease H: the enzymes in eukaryotes. FEBS J 276:1494-505
Chon, Hyongi; Vassilev, Alex; DePamphilis, Melvin L et al. (2009) Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex. Nucleic Acids Res 37:96-110
Rohman, Muhammad S; Koga, Yuichi; Takano, Kazufumi et al. (2008) Effect of the disease-causing mutations identified in human ribonuclease (RNase) H2 on the activities and stabilities of yeast RNase H2 and archaeal RNase HII. FEBS J 275:4836-49
Nowotny, Marcin; Cerritelli, Susana M; Ghirlando, Rodolfo et al. (2008) Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD. EMBO J 27:1172-81