UV damage regulates alternative polyadenylation of the RPB2 gene in yeast

Alternative polyadenylation (APA) is conserved in all eukaryotic cells. Selective use of polyadenylation sites appears to be a highly regulated process and contributes to human pathogenesis. In this article we report that the yeast RPB2 gene is alternatively polyadenylated, producing two mRNAs with different lengths of 3\u27UTR. In normally growing wild-type cells, polyadenylation preferentially uses the promoter-proximal poly(A) site. After UV damage transcription of RPB2 is initially inhibited. As transcription recovers, the promoter-distal poly(A) site is preferentially used instead, producing more of a longer form of RPB2 mRNA. We show that the relative increase in the long RPB2 mRNA is not caused by increased mRNA stability, supporting the preferential usage of the distal poly(A) site during transcription recovery. We demonstrate that the 3\u27UTR of RPB2 is sufficient for this UV-induced regulation of APA. We present evidence that while transcription initiation rates do not seem to influence selection of the poly(A) sites of RPB2, the rate of transcription elongation is an important determinant

Structural/functional analysis of the human OXR1 protein: identification of exon 8 as the anti-oxidant encoding function

BACKGROUND: The human OXR1 gene belongs to a class of genes with conserved functions that protect cells from reactive oxygen species (ROS). The gene was found using a screen of a human cDNA library by its ability to suppress the spontaneous mutator phenotype of an E. coli mutH nth strain. The function of OXR1 is unknown. The human and yeast genes are induced by oxidative stress and targeted to the mitochondria; the yeast gene is required for resistance to hydrogen peroxide. Multiple spliced isoforms are expressed in a variety of human tissues, including brain. RESULTS: In this report, we use a papillation assay that measures spontaneous mutagenesis of an E. coli mutM mutY strain, a host defective for oxidative DNA repair. Papillation frequencies with this strain are dependent upon a G-\u3eT transversion in the lacZ gene (a mutation known to occur as a result of oxidative damage) and are suppressed by in vivo expression of human OXR1. N-terminal, C-terminal and internal deletions of the OXR1 gene were constructed and tested for suppression of the mutagenic phenotype of the mutM mutY strain. We find that the TLDc domain, encoded by the final four exons of the OXR1 gene, is not required for papillation suppression in E. coli. Instead, we show that the protein segment encoded by exon 8 of OXR1 is responsible for the suppression of oxidative damage in E. coli. CONCLUSION: The protein segment encoded by OXR1 exon 8 plays an important role in the anti-oxidative function of the human OXR1 protein. This result suggests that the TLDc domain, found in OXR1 exons 12-16 and common in many proteins with nuclear function, has an alternate (undefined) role other than oxidative repair

Preventing Neurodegeneration by Controlling Oxidative Stress: The Role of OXR1

Parkinson’s disease, diabetic retinopathy, hyperoxia induced retinopathy, and neuronal damage resulting from ischemia are among the notable neurodegenerative diseases in which oxidative stress occurs shortly before the onset of neurodegeneration. A shared feature of these diseases is the depletion of OXR1 (oxidation resistance 1) gene products shortly before the onset of neurodegeneration. In animal models of these diseases, restoration of OXR1 has been shown to reduce or eliminate the deleterious effects of oxidative stress induced cell death, delay the onset of symptoms, and reduce overall severity. Moreover, increasing OXR1 expression in cells further increases oxidative stress resistance and delays onset of disease while showing no detectable side effects. Thus, restoring or increasing OXR1 function shows promise as a therapeutic for multiple neurodegenerative diseases. This review examines the role of OXR1 in oxidative stress resistance and its impact on neurodegenerative diseases. We describe the potential of OXR1 as a therapeutic in light of our current understanding of its function at the cellular and molecular level and propose a possible cascade of molecular events linked to OXR1’s regulatory functions

Gene expression caused by alkylating agents and cis-diamminedichloroplatinum(II) in Escherichia coli

Previous work has demonstrated heterogeneous effects of methylating agents on induction of DNA damage inducible genes in Escherichia coli. These studies employed E. coli mutants that have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents. These mutants were selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methylmethanesulfonate or N-methyl-N\u27-nitro-N-nitrosoguanidine. The current report extends these findings by analyzing gene expression caused by mechlorethamine, chloroethylnitrosoureas and cis-diamminedichloroplatinum(II) (cis-DDP). The results demonstrate heterogeneous effects by these agents on gene expression. While 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea induces alkA, other nitrosoureas, mechlorethamine, and cis-DDP do not cause expression of this gene. Further, while all nitrosoureas caused expression of aidC, mechlorethamine and cis-DDP did not. Lastly, cis-DDP caused marked expression of a sulA fusion mutant while not inducing any of the other E. coli fusion mutants

DNA double strand breaks but not interstrand crosslinks prevent progress through meiosis in fully grown mouse oocytes

There is some interest in how mammalian oocytes respond to different types of DNA damage because of the increasing expectation of fertility preservation in women undergoing chemotherapy. Double strand breaks (DSBs) induced by ionizing radiation and agents such as neocarzinostatin (NCS), and interstrand crosslinks (ICLs) induced by alkylating agents such as mitomycin C (MMC), are toxic DNA lesions that need to be repaired for cell survival. Here we examined the effects of NCS and MMC treatment on oocytes collected from antral follicles in mice, because potentially such oocytes are readily collected from ovaries and do not need to be in vitro grown to achieve meiotic competency. We found that oocytes were sensitive to NCS, such that this ionizing radiation mimetic blocked meiosis I and caused fragmented DNA. In contrast, MMC had no impact on the completion of either meiosis I or II, even at extremely high doses. However, oocytes treated with MMC did show ?-H2AX foci and following their in vitro maturation and parthenogenetic activation the development of the subsequent embryos was severely compromised. Addition of MMC to 1-cell embryos caused a similarly poor level of development, demonstrating oocytes have eventual sensitivity to this ICL-inducing agent but this does not occur during their meiotic division. In oocytes, the association of Fanconi Anemia protein, FANCD2, with sites of ICL lesions was not apparent until entry into the embryonic cell cycle. In conclusion, meiotic maturation of oocytes is sensitive to DSBs but not ICLs. The ability of oocytes to tolerate severe ICL damage and yet complete meiosis, means that this type of DNA lesion goes unrepaired in oocytes but impacts on subsequent embryo quality

RNA polymerase II depletion promotes transcription of alternative mRNA species

BACKGROUND: Cells respond to numerous internal and external stresses, such as heat, cold, oxidative stress, DNA damage, and osmotic pressure changes. In most cases, the primary response to stress is transcriptional induction of genes that assist the cells in tolerating the stress and facilitate the repair of the cellular damage. However, when the transcription machinery itself is stressed, responding by such standard mechanisms may not be possible. RESULTS: In this study, we demonstrate that depletion or inactivation of RNA polymerase II (RNAPII) changes the preferred polyadenylation site usage for several transcripts, and leads to increased transcription of a specific subset of genes. Surprisingly, depletion of RNA polymerase I (RNAPI) also promotes altered polyadenylation site usage, while depletion of RNA polymerase III (RNAPIII) does not appear to have an impact. CONCLUSIONS: Our results demonstrate that stressing the transcription machinery by depleting either RNAPI or RNAPII leads to a novel transcriptional response that results in induction of specific mRNAs and altered polyadenylation of many of the induced transcripts

The OXR domain defines a conserved family of eukaryotic oxidation resistance proteins

BACKGROUND: The NCOA7 gene product is an estrogen receptor associated protein that is highly similar to the human OXR1 gene product, which functions in oxidation resistance. OXR genes are conserved among all sequenced eukaryotes from yeast to humans. In this study we examine if NCOA7 has an oxidation resistance function similar to that demonstrated for OXR1. We also examine NCOA7 expression in response to oxidative stress and its subcellular localization in human cells, comparing these properties with those of OXR1. RESULTS: We find that NCOA7, like OXR1 can suppress the oxidative mutator phenotype when expressed in an E. coli strain that exhibits an oxidation specific mutator phenotype. Moreover, NCOA7's oxidation resistance function requires expression of only its carboxyl-terminal domain and is similar in this regard to OXR1. We find that, in human cells, NCOA7 is constitutively expressed and is not induced by oxidative stress and appears to localize to the nucleus following estradiol stimulation. These properties of NCOA7 are in striking contrast to those of OXR1, which is induced by oxidative stress, localizes to mitochondria, and appears to be excluded, or largely absent from nuclei. CONCLUSION: NCOA7 most likely arose from duplication. Like its homologue, OXR1, it is capable of reducing the DNA damaging effects of reactive oxygen species when expressed in bacteria, indicating the protein has an activity that can contribute to oxidation resistance. Unlike OXR1, it appears to localize to nuclei and interacts with the estrogen receptor. This raises the possibility that NCOA7 encodes the nuclear counterpart of the mitochondrial OXR1 protein and in mammalian cells it may reduce the oxidative by-products of estrogen metabolite-mediated DNA damage

Molecular Assessment of Epiretinal Membrane: Activated Microglia, Oxidative Stress and Inflammation

Fibrocellular membrane or epiretinal membrane (ERM) forms on the surface of the inner limiting membrane (ILM) in the inner retina and alters the structure and function of the retina. ERM formation is frequently observed in ocular inflammatory conditions, such as proliferative diabetic retinopathy (PDR) and retinal detachment (RD). Although peeling of the ERM is used as a surgical intervention, it can inadvertently distort the retina. Our goal is to design alternative strategies to tackle ERMs. As a first step, we sought to determine the composition of the ERMs by identifying the constituent cell-types and gene expression signature in patient samples. Using ultrastructural microscopy and immunofluorescence analyses, we found activated microglia, astrocytes, and Muller glia in the ERMs from PDR and RD patients. Moreover, oxidative stress and inflammation associated gene expression was significantly higher in the RD and PDR membranes as compared to the macular hole samples, which are not associated with inflammation. We specifically detected differential expression of hypoxia inducible factor 1-alpha (HIF1-alpha), proinflammatory cytokines, and Notch, Wnt, and ERK signaling pathway-associated genes in the RD and PDR samples. Taken together, our results provide new information to potentially develop methods to tackle ERM formation

Flavin-Induced Oligomerization in Escherichia coli Adaptive Response Protein AidB

The process known as “adaptive response” allows Escherichia coli to respond to small doses of DNA-methylating agents by upregulating the expression of four proteins. While the role of three of these proteins in mitigating DNA damage is well understood, the function of AidB is less clear. Although AidB is a flavoprotein, no catalytic role has been established for the bound cofactor. Here we investigate the possibility that flavin plays a structural role in the assembly of the AidB tetramer. We report the generation and biophysical characterization of deflavinated AidB and of an AidB mutant that has greatly reduced affinity for flavin adenine dinucleotide (FAD). Using fluorescence quenching and analytical ultracentrifugation, we find that apo AidB has a high affinity for FAD, as indicated by an apparent dissociation constant of 402.1 ± 35.1 nM, and that binding of substoichiometric amounts of FAD triggers a transition in the AidB oligomeric state. In particular, deflavinated AidB is dimeric, whereas the addition of FAD yields a tetramer. We further investigate the dimerization and tetramerization interfaces of AidB by determining a 2.8 Å resolution crystal structure in space group P32 that contains three intact tetramers in the asymmetric unit. Taken together, our findings provide strong evidence that FAD plays a structural role in the formation of tetrameric AidB.National Institutes of Health (U.S.) (grant R01-GM0272663)National Institutes of Health (U.S.) (grant P30-ES002109)National Science Foundation (U.S.) (grant MCB-0543833