Evidence of the modulation of mRNA splicing fidelity in humans by oxidative stress and p53.

The majority of human genes generate mRNA splice variants and while there is little doubt that alternative splicing is an important biological phenomenon, it has also become apparent that some splice variants are associated with disease. To elucidate the molecular mechanisms responsible for generating aberrant splice variants, we have investigated alternative splicing of the human genes HPRT and POLB following oxidative stress in different genetic backgrounds. Our study revealed that splicing fidelity is sensitive to oxidative stress. Following treatment of cells with H2O2, the overall frequency of aberrant, unproductive splice variants increased in both loci. At least in POLB, splicing fidelity is p53 dependent. In the absence of p53, the frequency of POLB splice variants is elevated but oxidative stress does not further increase the frequency of splice variants. Our data indicate that mis-splicing following oxidative stress represents a novel and significant genotoxic outcome and that it is not simply DNA lesions induced by oxidative stress that lead to mis-splicing but changes in the alternative splicing machinery itself.     

Splicing of SV40 early pre-mRNA to large T and small t mRNAs utilizes different patterns of lariat branch sites

To explore the mechanism and control of alternative splicing, we have characterized the products formed by splicing of SV40 early pre-mRNA in vitro and in vivo. Large T and small t mRNAs are derived from this precursor by joining alternative 5' splice sites to a single shared 3' splice site. In contrast to pre-mRNAs studied previously, we have shown that splicing to large T RNA involves the utilization of multiple lariat branch sites, while small t splicing uses a single branch site. Interestingly, the predominant branch sites utilized in splicing of large T RNA in vitro were found to differ in nuclear extracts from HeLa and human 293 cells, correlated with previously observed differences in the ratio of large T to small t mRNAs produced in the two cell types. To test the significance of this correlation, we examined the products formed by splicing of an SV40 early precursor microinjected into X. laevis oocytes. Strikingly, both the pattern of branch sites used in large T splicing and the ratio of large T to small t mRNAs produced were found to be identical to those observed in 293 cells and extracts.     

Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase

The free radical hypothesis of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species that are produced as by-products of normal metabolic processes. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic (IDPc) and mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate that modulation of IDPc or IDPm activity in IMR-90 cells regulates cellular redox status and replicative senescence. When we examined the regulatory role of IDPc and IDPm against the aging process with IMR-90 cells transfected with cDNA for IDPc or IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc or IDPm expressed in target cells and their susceptibility to senescence, which was reflected by changes in replicative potential, cell cycle, senescence-associated beta-galactosidase activity, expression of p21 and p53, and morphology of cells. Furthermore, lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher and cellular redox status shifted to a prooxidant condition in the cell lines expressing the lower level of IDPc or IDPm. The results suggest that IDPc and IDPm play an important regulatory role in cellular defense against oxidative stress and in the senescence of IMR-90 cells.     

Oxidative stress in human aging and mitochondrial disease-consequences of defective mitochondrial respiration and impaired antioxidant enzyme system

Respiratory function of mitochondria is compromised in aging human tissues and severely impaired in the patients with mitochondrial disease. A wide spectrum of mitochondrial DNA (mtDNA) mutations has been established to associate with mitochondrial diseases. Some of these mtDNA mutations also occur in various human tissues in an age-dependent manner. These mtDNA mutations cause defects in the respiratory chain due to impairment of the gene expression and structure of respiratory chain polypeptides that are encoded by the mitochondrial genome. Since defective mitochondria generate more reactive oxygen species (ROS) such as O2- and H2O2 via electron leak, we hypothesized that oxidative stress is a contributory factor for aging and mitochondrial disease. This hypothesis has been supported by the findings that oxidative stress and oxidative damage in tissues and culture cells are increased in elderly subjects and patients with mitochondrial diseases. Another line of supporting evidence is our recent finding that the enzyme activities of Cu,Zn-SOD, catalase and glutathione peroxidase (GPx) decrease with age in skin fibroblasts. By contrast, Mn-SOD activity increases up to 65 years of age and then slightly declines thereafter. On the other hand, we observed that the RNA, protein and activity levels of Mn-SOD are increased two- to three-fold in skin fibroblasts of the patients with CPEO syndrome but are dramatically decreased in patients with MELAS or MERRF syndrome. However, the other antioxidant enzymes did not change in the same manner. The imbalance in the expression of these antioxidant enzymes indicates that the production of ROS is in excess of their removal, which in turn may elicit an elevation of oxidative stress in the fibroblasts. Indeed, it was found that intracellular levels of H2O2 and oxidative damage to DNA and lipids in skin fibroblasts from elderly subjects or patients with mitochondrial diseases are significantly increased as compared to those of age-matched controls. Furthermore, Mn-SOD or GPx-1 gene knockout mice were found to display neurological disorders and enhanced oxidative damage similar to those observed in the patients with mitochondrial disease. These observations are reviewed in this article to support that oxidative stress elicited by defective respiratory function and impaired antioxidant enzyme system plays a key role in the pathophysiology of mitochondrial disease and human aging.     

Endogenous mitochondrial oxidative stress: neurodegeneration, proteomic analysis, specific respiratory chain defects, and efficacious antioxidant therapy in superoxide dismutase 2 null mice

Oxidative stress and mitochondrial dysfunction have been linked to neurodegenerative disorders such as Parkinson's and Alzheimer's disease. However, it is not yet understood how endogenous mitochondrial oxidative stress may result in mitochondrial dysfunction. Most prior studies have tested oxidative stress paradigms in mitochondria through either chemical inhibition of specific components of the respiratory chain, or adding an exogenous insult such as hydrogen peroxide or paraquat to directly damage mitochondria. In contrast, mice that lack mitochondrial superoxide dismutase (SOD2 null mice) represent a model of endogenous oxidative stress. SOD2 null mice develop a severe neurological phenotype that includes behavioral defects, a severe spongiform encephalopathy, and a decrease in mitochondrial aconitase activity. We tested the hypothesis that specific components of the respiratory chain in the brain were differentially sensitive to mitochondrial oxidative stress, and whether such sensitivity would lead to neuronal cell death. We carried out proteomic differential display and examined the activities of respiratory chain complexes I, II, III, IV, V, and the tricarboxylic acid cycle enzymes alpha-ketoglutarate dehydrogenase and citrate synthase in SOD2 null mice in conjunction with efficacious antioxidant treatment and observed differential sensitivities of mitochondrial proteins to oxidative stress. In addition, we observed a striking pattern of neuronal cell death as a result of mitochondrial oxidative stress, and were able to significantly reduce the loss of neurons via antioxidant treatment.     

Short‐term ammonium supply induces cellular defence to prevent oxidative stress in Arabidopsis leaves

Plants can assimilate nitrogen from soil pools of both ammonium and nitrate, and the relative levels of these two nitrogen sources are highly variable in soil. Long‐term ammonium nutrition is known to cause damage to Arabidopsis that has been linked to mitochondrial oxidative stress. Using hydroponic cultures, we analysed the consequences of rapid shifts between nitrate and ammonium nutrition. This did not induce growth retardation, showing that Arabidopsis can compensate for the changes in redox metabolism associated with the variations in nitrogen redox status. During the first 3 h of ammonium treatment, we observed distinct transient shifts in reactive oxygen species (ROS), low‐mass antioxidants, ROS‐scavenging enzymes, and mitochondrial alternative electron transport pathways, indicating rapid but temporally separated changes in chloroplastic, mitochondrial and cytosolic ROS metabolism. The fast induction of antioxidant defences significantly lowered intracellular H₂O₂ levels, and thus protected Arabidopsis leaves from oxidative stress. On the other hand elevated extracellular ROS production in response to ammonium supply may be involved in signalling. The response pattern displays an intricate plasticity of Arabidopsis redox metabolism to minimise stress in responses to nutrient changes.     

The mitochondrial antioxidant defence system and its response to oxidative stress

The antioxidant systems of mitochondria are not well known. Using a proteomics-based approach, we defined these mitochondrial antioxidant systems and analyzed their response to oxidative stress. It appears that the major mitochondrial antioxidant system is made of manganese superoxide dismutase on the one hand, and of peroxiredoxin III, mitochondrial thioredoxin and mitochondrial thioredoxin reductase on the other hand. With the exception of thioredoxin reductase, all these proteins are induced by oxidative stress. In addition, a change in the peroxiredoxin III pattern can also be observed.     

Regulation and function of ascorbate peroxidase isoenzymes

Even under optimal conditions, many metabolic processes, including the chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems of higher plants, produce active oxygen species (AOS). Furthermore, the imposition of biotic and abiotic stress conditions can give rise to excess concentrations of AOS, resulting in oxidative damage at the cellular level. Therefore, antioxidants and antioxidant enzymes function to interrupt the cascades of uncontrolled oxidation in each organelle. Ascorbate peroxidase (APX) exists as isoenzymes and plays an important role in the metabolism of H(2)O(2) in higher plants. APX is also found in eukaryotic algae. The characterization of APX isoenzymes and the sequence analysis of their clones have led to a number of investigations that have yielded interesting and novel information on these enzymes. Interestingly, APX isoenzymes of chloroplasts in higher plants are encoded by only one gene, and their mRNAs are generated by alternative splicing of the gene's two 3'-terminal exons. Manipulation of the expression of the enzymes involved in the AOS-scavenging systems by gene-transfer technology has provided a powerful tool for increasing the present understanding of the potential of the defence network against oxidative damage caused by environmental stresses. Transgenic plants expressing E. coli catalase to chloroplasts with increased tolerance to oxidative stress indicate that AOS-scavenging enzymes, especially chloroplastic APX isoenzymes are sensitive under oxidative stress conditions. It is clear that a high level of endogenous ascorbate is essential effectively to maintain the antioxidant system that protects plants from oxidative damage due to biotic and abiotic stresses.     

Phosphorylation of SRSF1 is modulated by replicational stress

DNA ligase I-deficient 46BR.1G1 cells show a delay in the maturation of replicative intermediates resulting in the accumulation of single- and double-stranded DNA breaks. As a consequence the ataxia telangiectasia mutated protein kinase (ATM) is constitutively phosphorylated at a basal level. Here, we use 46BR.1G1 cells as a model system to study the cell response to chronic replication-dependent DNA damage. Starting from a proteomic approach, we demonstrate that the phosphorylation level of factors controlling constitutive and alternative splicing is affected by the damage elicited by DNA ligase I deficiency. In particular, we show that SRSF1 is hyperphosphorylated in 46BR.1G1 cells compared to control fibroblasts. This hyperphosphorylation can be partially prevented by inhibiting ATM activity with caffeine. Notably, hyperphosphorylation of SRSF1 affects the subnuclear distribution of the protein and the alternative splicing pattern of target genes. We also unveil a modulation of SRSF1 phosphorylation after exposure of MRC-5V1 control fibroblasts to different exogenous sources of DNA damage. Altogether, our observations indicate that a relevant aspect of the cell response to DNA damage involves the post-translational regulation of splicing factor SRSF1 which is associated with a shift in the alternative splicing program of target genes to control cell survival or cell death.     

Paraquat Modulates Alternative Pre-mRNA Splicing by Modifying the Intracellular Distribution of SRPK2

Paraquat (PQ) is a neurotoxic herbicide that induces superoxide formation. Although it is known that its toxic properties are linked to ROS production, the cellular response to PQ is still poorly understood. We reported previously that treatment with PQ induced genome-wide changes in pre-mRNA splicing. Here, we investigated the molecular mechanism underlying PQ-induced pre-mRNA splicing alterations. We show that PQ treatment leads to the phosphorylation and nuclear accumulation of SRPK2, a member of the family of serine/arginine (SR) protein-specific kinases. Concomitantly, we observed increased phosphorylation of SR proteins. Site-specific mutagenesis identified a single serine residue that is necessary and sufficient for nuclear localization of SRPK2. Transfection of a phosphomimetic mutant modified splice site selection of the E1A minigene splicing reporter similar to PQ-treatment. Finally, we found that PQ induces DNA damage and vice versa that genotoxic treatments are also able to promote SRPK2 phosphorylation and nuclear localization. Consistent with these observations, treatment with PQ, cisplatin or γ-radiation promote changes in the splicing pattern of genes involved in DNA repair, cell cycle control, and apoptosis. Altogether, our findings reveal a novel regulatory mechanism that connects PQ to the DNA damage response and to the modulation of alternative splicing via SRPK2 phosphorylation.     

Mitochondrial dysfunction in cardiovascular disease

Whereas the pathogenesis of atherosclerosis has been intensively studied and described, the underlying events that initiate cardiovascular disease are not yet fully understood. A substantial number of studies suggest that altered levels of oxidative and nitrosoxidative stress within the cardiovascular environment are essential in the development of cardiovascular disease; however, the impact of such changes on the subcellular or organellar components and their functions that are relevant to cardiovascular disease inception are less understood. In this regard, studies are beginning to show that mitochondria not only appear susceptible to damage mediated by increased oxidative and nitrosoxidative stress, but also play significant roles in the regulation of cardiovascular cell function. In addition, accumulating evidence suggests that a common theme among cardiovascular disease development and cardiovascular disease risk factors is increased mitochondrial damage and dysfunction. This review discusses aspects relating mitochondrial damage and function to cardiovascular disease risk factors and disease development.     

Dietary restriction attenuates age-associated muscle atrophy by lowering oxidative stress in mice even in complete absence of CuZnSOD

Age-related loss of muscle mass and function, sarcopenia, has a major impact on the quality of life in the elderly. Among the proposed causes of sarcopenia are mitochondrial dysfunction and accumulated oxidative damage during aging. Dietary restriction (DR), a robust dietary intervention that extends lifespan and modulates age-related pathology in a variety of species, has been shown to protect from sarcopenia in rodents. Although the mechanism(s) by which DR modulates aging are still not defined, one potential mechanism is through modulation of oxidative stress and mitochondrial dysfunction. To directly test the protective effect of DR against oxidative stress-induced muscle atrophy in vivo, we subjected mice lacking a key antioxidant enzyme, CuZnSOD (Sod1) to DR (60% of ad libitum fed diet). We have previously shown that the Sod1(-/-) mice exhibit an acceleration of sarcopenia associated with high oxidative stress, mitochondrial dysfunction, and severe neuromuscular innervation defects. Despite the dramatic atrophy phenotype in the Sod1(-/-) mice, DR led to a reversal or attenuation of reduced muscle function, loss of innervation, and muscle atrophy in these mice. DR improves mitochondrial function as evidenced by enhanced Ca(2+) regulation and reduction of mitochondrial reactive oxygen species (ROS). Furthermore, we show upregulation of SIRT3 and MnSOD in DR animals, consistent with reduced mitochondrial oxidative stress and reduced oxidative damage in muscle tissue measured as F(2) -isoprostanes. Collectively, our results demonstrate that DR is a powerful mediator of mitochondrial function, mitochondrial ROS production, and oxidative damage, providing a solid protection against oxidative stress-induced neuromuscular defects and muscle atrophy in vivo even under conditions of high oxidative stress.     

Manganese superoxide dismutase induction by iron is impaired in Friedreich ataxia cells.

Iron-mediated oxidative stress has been implicated in the pathology of the neurodegenerative disease Friedreich ataxia (FRDA). Here, we show that normal upregulation of the stress defense protein manganese superoxide dismutase (MnSOD) fails to occur in FRDA fibroblasts exposed to iron. This impaired induction was observed at iron levels in which increased activation of the redox-sensitive factor NF-kappaB was absent. Furthermore, MnSOD induction could only be partially suppressed by antioxidants. We conclude that an NF-kappaB-independent pathway that may not require free radical signaling is responsible for the reduction of MnSOD induction. This impairment could constitute both a novel defense mechanism against iron-mediated oxidative stress in cells with mitochondrial iron overload and conversely, an alternative source of free radicals that could contribute to the disease pathology.     

SC35 autoregulates its expression by promoting splicing events that destabilize its mRNAs

SC35 belongs to the family of SR proteins that regulate alternative splicing in a concentration-dependent manner in vitro and in vivo. We previously reported that SC35 is expressed through alternatively spliced mRNAs with differing 3' untranslated sequences and stabilities. Here, we show that overexpression of SC35 in HeLa cells results in a significant decrease of endogenous SC35 mRNA levels along with changes in the relative abundance of SC35 alternatively spliced mRNAs. Remarkably, SC35 leads to both an exon inclusion and an intron excision in the 3' untranslated region of its mRNAs. In vitro splicing experiments performed with recombinant SR proteins demonstrate that SC35, but not ASF/SF2 or 9G8, specifically activates these alternative splicing events. Interestingly, the resulting mRNA is very unstable and we present evidence that mRNA surveillance is likely to be involved in this instability. SC35 therefore constitutes the first example of a splicing factor that controls its own expression through activation of splicing events leading to expression of unstable mRNA.     

Patterns of alternative splicing of fibronectin pre-mRNA in human adult and fetal tissues

Alternative splicing of fibronectin pre-mRNA at two distinct regions, termed ED-A and IIICS, was investigated with human adult and fetal tissues by the nuclease S1 protection assay. A clear tissue specificity was observed in the splicing pattern at the ED-A region. More ED-A+ than ED-A- mRNAs were identified in lung, whereas ED-A- mRNAs were predominantly expressed in liver. Endometrium contained nearly equal amounts of ED-A+ and ED-A- mRNAs. The splicing pattern at the ED-A region was also different between adult and fetal liver but not between adult and fetal lung. Tissue type specific splicing was also observed at the IIICS region. Although the mRNA species containing the complete IIICS sequence comprised 40-65% of the total fibronectin mRNAs irrespective of tissue types, expression of the mRNA species lacking a part or all of the IIICS sequence was more pronounced in adult liver than in other tissues including fetal liver. These results strongly suggest that the alternative splicing of fibronectin pre-mRNA in vivo is regulated in a tissue type specific manner at both the ED-A and IIICS regions and that it is developmentally regulated in liver but not in lung. On the basis of these and other observations reported previously, a possibility that a part of the fibronectins synthesized and secreted by hepatocytes is deposited in the tissue matrix is discussed.