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 then 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. A mouse model of Lissencephaly5 R-loops are three stranded structures in which RNA loops out a DNA strand by annealing to the non-displaced DNA strand. It has been proposed that triplet expansion disease could result from improper realigning when the RNA is removed. We were excited to see a mouse with severe ataxia appear in a mating with mice bearing mutations in the Rnaseh1 gene and wanted to understand the origin of the phenotype in this mouse. A point mutation in the Lamb1 gene rather than a triplet-expansion disease was discovered as the cause for the cerebellar ataxia. Mutations in LAMB1 of human patients are known causes of Lissencephaly5, a neuronal migration disorder with characteristic protrusions of neurons. Lamb1 is a part of a complex of three similar proteins, a1, b1 and c1 that associate with dystroglycans and are part of congenital muscular dystrophies. One model for assembly of the complexes posits that these basement membrane proteins interact in an umbrella-like manner with the N-terminal domain of each protein interacting with one or more adjacent a1,b1,c1 complexes forming an external canopy. The coiled-coiled region forms a handle of the umbrella. The mutation we found in Lamb1 is in the N-terminal loop of Lamb1 where an exposed serine has been substituted with a leucine. Structural studies could be used to support a role for the ser in protein-protein interactions. We performed multiple H&E staining and immunofluorescent studies which showed abnormal cerebellar lobe folding when the Lamb1Ser-Leu mutation was homozygous but not when heterozygous. Lobes VI and VII were the most disordered with an unusual area, possibly associated with the fastigial nucleus and/or poorly migrated components of lobes VI and VII. Decreased Lamb1 immunofluorescence signal was seen throughout the cerebellar sections in both Lamb1-hets and homozygous mutants. The structural integrity of the basement membrane and a normal gate can be maintained with what appears to be 50% of normal a1,b1,c1 laminin. We expect this interesting mouse to become a model for human Lissencephaly5. Increased incorporation of ribonucleotides in yeast DNA leads to genomic instability in the absence of RNases H The cellular concentration of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA, are tightly controlled during the cell cycle to allow the replication and repair of genomic DNA. Ribonucleotide reductase (RNR) is a key enzyme which converts ribonucleotides to deoxyribonucleotides and regulates dNTP/rNTP ratios. Cellular levels of dNTPs are much lower than those of rNTPs but increase when cells enter S-phase to facilitate genomic DNA synthesis. In addition, DNA Polymerases (Pol) have active sites that select against the bulky rNTP residues in favor of dNTPs, which fit much better. To increase the incorporation of rNMPs in the DNA of Saccharomyces cerevisiae, we reduced the dNTP pools by depleting Rnr1, the major catalytic subunit of RNR. We observed that when dNTP pools were decreased, RNase H1 and RNase H2 were required for viability. We suggested that under the conditions of replicative stress created by low dNTP concentrations, R-loops that accumulate in the absence of both RNase H1 and RNase H2 could become insurmountable impediments to the progression of the replication fork, inducing fork collapse and genomic instability. We further increased rNMPs incorporation in genomic DNA by depleting Rnr1 in strains that harbor DNA Pol mutants that have higher propensity to incorporate rNMPs in genomic DNA. In these conditions, RNase H2 became essential. In addition to R-loops processing, RNase H2-initiates the Ribonucleotide Excision Repair process, which efficiently removes single rNMPs in genomic DNA. The lethality of the triple mutant lacking RNase H2 in the DNA Pol mutant depleted of Rnr1, could be reversed in the absence of Topoisomerase (Top1). We concluded that when RER is defective, Top1 processes rNMPs in DNA in a mutagenic way, inducing genome instability. When a threshold of single genomic rNMPs is exceeded in cells with limited dNTP pools and absence of RER, the Top1-mediated DNA damage results in severe growth defects and lethality.