Factors Influencing Genetic Transcription Initiation And Termination

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Loss of RNase H1 in early B cell development activates mitochondrial Unfolded Protein Response without affecting the nuclear R-loops We made a knockout of the mouse Rnaseh1 gene and discovered that two proteins of RNase H1 are produced from a single mRNA by a leaky scanning method for differential translation. One protein is localized to the nucleus and a second is targeted to mitochondria. Nuclear DNA replication begins at fertilization with mitochondrial DNA beginning amplification several days later. We observed early embryonic death shortly after mtDNA should have begun, thereby linking absence of mtDNA replication and death. We were curious to see the contribution effects in a system less complicated than embryonic development. We chose mouse B cell development because i) B cells are not required for viability when mice are housed in a germ-free environment, ii) B cell development occurs in only a few rounds of cell duplication, iii) resting B cells are in G0 providing a population of cells that respond together when stimulated, iv) many useful tools for analyses and manipulation are available and v) RNase H has potential known substrates in B cell development. We generated an Rnaseh1 conditional KO mouse strain in which we can specifically KO the gene using a CRE-lox method with the mb1 promoter driving CRE. Transcripts of mb1 are initiated from the earliest stage of B cell and persist until plasmacytes are formed. B cells develop to the resting stage at which point they can be stimulated to undergo isotype switching by class switch recombination (CSR), ultimately producing circulating antibodies. We found that mb1-CRE KO of the Rnaseh1 gene resulted in little or no circulating antibodies but did produce resting B cells, although yielding half as many B cells as WT mice. Stimulation of these B cells initiated transitioning from G0 to G1 phase of the cell cycle, but essentially never entered S-phase. The resting B cells had no intact Rnaseh1 gene, no mtDNA, cells had no RNase H1 activity and mitochondria exhibited abnormal morphology. RNA-seq analyses of resting and 24 h-stimulated mutant and WT B cells was performed to discover genes related to loss of mtDNA and/or a nuclear DNA damage response. Pathways that exhibited decreases were Cell Cycle, immune system, DNA replication, mitochondrion, RNA processing and ribosomes. The 50% yield of resting B cell in the KO strain must occur during cell amplification in bone marrow. The loss of RNase H1 was initiated just prior to cell amplification and might limit the number of cell cycles. It is also possible that defects affecting the time of residence of the B cells in the bone marrow niche are affected. Loss of the Nidogen1 gene results in reduction of resting B cells to 50% normal, the same as our KO mice. We noticed a significant difference between WT and mutant resting B cells for the Nidogen1 transcripts. The list of genes with the highest fold difference between resting and stimulated KO mice are Atf5, Gdf15, Atf3, Hspa9, and Ddit3. Atf5, Atf3 and Ddit3 all of which are hallmarks of the Unfolded Mitochondrial Response (UPRmt). The activation of the UPRmt indicates that loss of mtDNA takes precedence over are nuclear DNA damage response, just as we observed in embryonic development when the Rnaseh1 gene was deleted in the male and female gametes. We checked the presence of R-loops by DRIP-seq and surprisingly found no alteration in R-loops indicating the lack of RNase H1 in processing these structures. These results indicate that the loss of mitochondrial function masks any contribution of the role played by the nuclear form of RNase H1. To gain more insight into the role of the nuclear form of the enzyme, we will generate a mouse that produces only the mitochondrial form. If viable, we will observe differences, if any, in growth, fecundity, longevity and challenges to DNA damage. If embryonic lethal, we will examine embryonic day of lethality and examine differences between normal and mutated mice at that date. RNase H2 and Aicardi-Goutires Syndrome We have continued our studies on the Rnaseh2aG37S (G37S) mouse model of Aicardi-Goutires Syndrome. Homozygous (G37S/G37S) mice are obtained at non-Mendelian frequencies and exhibit several phenotypes including small size. We previously found that homozygosity activated the cGAS-Sting innate immune pathway a dsDNA dependent mechanism. Our current model for the source of the activating DNA results from double-stranded breaks, most likely derived from R-loops whose presence are mostly observed in ribosomal DNA. RNases H and the RNA exosome and cooperate in suppressing R-loop-mediated genome instability RNA transcripts can engage co- or post-transcriptionally with the template DNA strand, forming an RNA/DNA hybrid and leaving an unpaired single DNA strand, thus creating an R-loop. These structures can have physiological or pathological roles, and there are multiple mechanisms to resolve them and/or prevent their formation. Unscheduled and/or persistent R-loops can lead to gene expression defects, transcription-replication conflicts, replicative stress, and/or genome instability. We are studying the functional interactions between RNases H and the RNA exosome in preventing R-loop-mediated genome instability in budding yeast. Eukaryotic RNase H1 and RNase H2 (RNases H) cleave the RNA component of RNA/DNA hybrids, therefore eliminating R-loops. The RNA exosome is a major 3-5 RNA degradation and processing multi-subunit complex in eukaryotes, which, by removing cryptic and/or defective transcripts, would resolve R-loops and/or prevent their formation. Wild-type cells are sensitive to hydroxyurea, which induces replicative stress by decreasing the cellular dNTP supply. Interestingly, we found that cells defective for both RNases H and exosome activities are hypersensitive to hydroxyurea. This suggests that combined RNases H and exosome activities suppress R-loops that would otherwise exacerbate replicative stress in presence of low dNTP pools. We also found that RNase H1 over-expression partially suppresses the growth defects of exosome-deficient mutants, strongly suggesting that harmful R-loops can be prevented and/or eliminated by the exosome. To further elucidate the interplay between RNases H and the exosome in preventing genome instability, we are using a genetic system that correlates R-loop accumulation with homologous recombination (Kim and Jinks-Robertson, DNA Repair, 2011). We found that in mutants that are defective for both exosome and RNases H activities, there is both increased RNA accumulation and R-loop formation at the construct. These correlate well with a high increase in recombination rates. We conclude that RNases H and the exosome pathways converge to promote genome stability by suppressing the harmful effects of R-loops.