Tricarboxylic acid cycle activity suppresses acetylation of mitochondrial proteins during early embryonic development in Caenorhabditis elegans.

The tricarboxylic acid (TCA) cycle (or citric acid cycle) is responsible for the complete oxidation of acetyl-CoA and formation of intermediates required for ATP production and other anabolic pathways, such as amino acid synthesis. Here, we uncovered an additional mechanism that may help explain the essential role of the TCA cycle in the early embryogenesis of Caenorhabditis elegans. We found that knockdown of citrate synthase (cts-1), the initial and rate-limiting enzyme of the TCA cycle, results in early embryonic arrest, but that this phenotype is not because of ATP and amino acid depletions. As a possible alternative mechanism explaining this developmental deficiency, we observed that cts-1 RNAi embryos had elevated levels of intracellular acetyl-CoA, the starting metabolite of the TCA cycle. Of note, we further discovered that these embryos exhibit hyperacetylation of mitochondrial proteins. We found that supplementation with acetylase-inhibiting polyamines, including spermidine and putrescine, counteracted the protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Contrary to the hypothesis that spermidine acts as an acetyl sink for elevated acetyl-CoA, the levels of three forms of acetylspermidine, N1-acetylspermidine, N8-acetylspermidine, and N1,N8-diacetylspermidine, were not significantly increased in embryos treated with exogenous spermidine. Instead, we demonstrated that the mitochondrial deacetylase sirtuin 4 (encoded by the sir-2.2 gene) is required for spermidine's suppression of protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Taken together, these results suggest the possibility that during early embryogenesis, acetyl-CoA consumption by the TCA cycle in C. elegans prevents protein hyperacetylation and thereby protects mitochondrial function.

The GATA transcription factor ELT-2 modulates both the expression and methyltransferase activity of PRMT-1 in Caenorhabditis elegans.

Protein arginine methyltransferase 1 (PRMT1) catalyzes asymmetric arginine dimethylation of cellular proteins and thus modulates various biological processes, including gene regulation, RNA metabolism, cell signaling and DNA repair. Since prmt-1 null mutant completely abolishes asymmetric dimethylarginine in C. elegans, PRMT-1 is thought to play a crucial role in determining levels of asymmetric arginine dimethylation. However, the mechanism underlying the regulation of PRMT-1 activity remains largely unknown. Here, we explored for transcription factors that induce the expression of PRMT-1 by an RNAi screen using transgenic C. elegans harbouring prmt-1 promoter upstream of gfp. Of 529 clones, we identify a GATA transcription factor elt-2 as a positive regulator of Pprmt-1:: gfp expression and show that elt-2 RNAi decreases endogenous PRMT-1 expression at mRNA and protein levels. Nevertheless, surprisingly arginine methylation levels are increased when elt-2 is silenced, implying that erythroid-like transcription factor (ELT)-2 may also have ability to inhibit methyltransferase activity of PRMT-1. Supporting this idea, GST pull-down and co-immunoprecipitation assays demonstrate the interaction between ELT-2 and PRMT-1. Furthermore, we find that ELT-2 interferes with PRMT-1-induced arginine methylation in a dose-dependent manner. Collectively, our results illustrate the two modes of PRMT-1 regulation, which could determine the levels of asymmetric arginine dimethylation in C. elegans.

rRNA adenine methylation requires T07A9.8 gene as rram-1 in Caenorhabditis elegans.

RNAs are post-transcriptionally modified in all kingdoms of life. Of these modifications, base methylations are highly conserved in eukaryote ribosomal RNA (rRNA). Recently, rRNA processing protein 8 (Rrp8) and nucleomethylin (NML) were identified as factors of N1-methyladenosine (m1A) modification in yeast 25 S and mammalian 28 S rRNA, respectively. However, m1A modification of rRNA is still poorly understood in Caenorhabditis elegans (C. elegans). Here, using the liquid chromatography/tandem mass spectrometry analysis and RNA immunoprecipitation assay, we have identified that the m1A modification is located around position 674 (A674) of 26 S rRNA in C. elegans. Furthermore, quantitative PCR-based analysis revealed that T07A9.8, a C. elegans homolog of yeast Rrp8 and human NML, is responsible for m1A modification at A674 of 26 S rRNA. This m1A modification site in C. elegans corresponds to those in yeast 25 S rRNA and human 28 S rRNA. Intriguingly, T07A9.8 is not associated with pre-rRNA transcription under normal nutrient conditions. Since the m1A modification of 26 S rRNA requires T07A9.8 in C. elegans, we designated the gene as rRNA adenine methyltransferase-1 (rram-1).

The possible repositioning of an oral anti-arthritic drug, auranofin, for Nrf2-activating therapy: The demonstration of Nrf2-dependent anti-oxidative action using a zebrafish model.

The Nrf2 pathway is a biological defense system against oxidative stress. The pharmacological activation of the Nrf2 pathway is a promising therapy for oxidative stress-related diseases, but it has been challenging to find an Nrf2 activator with acceptable toxicity. To circumvent this problem, we focused on an already approved oral anti-arthritic drug, auranofin that has been reported to have the potential to activate Nrf2. We used a zebrafish model to investigate whether auranofin has protective action against oxidative stress in vivo. Auranofin pre-treatment considerably improved the survival of zebrafish larvae that were challenged with a lethal dose of hydrogen peroxide. This protective effect was not observed in an Nrf2 mutant zebrafish strain, suggesting that the activation of the biological defense against oxidative stress was Nrf2-dependent. Auranofin-induced protection was further tested by challenges with redox-active heavy metals. A clear protective effect was observed against arsenite, a highly redox-reactive toxicant. In addition, this effect was also demonstrated to be Nrf2-dependent based on the analysis of an Nrf2 mutant strain. These results clearly demonstrate the anti-oxidative action of auranofin and encourage the repositioning of auranofin as a drug that improves oxidative stress-related pathology.

Simultaneous ablation of prmt-1 and prmt-5 abolishes asymmetric and symmetric arginine dimethylations in Caenorhabditis elegans.

Protein arginine methyltransferases (PRMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to arginine residues and are classified into two types: type I producing asymmetric dimethylarginine (ADMA) and type II producing symmetric dimethylarginine (SDMA). PRMTs have been shown to regulate many cellular processes, including signal transduction, transcriptional regulation and RNA processing. Since the loss-of-function mutation of PRMT1 and PRMT5, each of which is the predominant type I and II, respectively, causes embryonic lethality in mice, their physiological significance at the whole-body level remains largely unknown. Here, we show the morphological and functional phenotypes of single or double null alleles of prmt-1 and prmt-5 in Caenorhabditis elegans. The prmt-1;prmt-5 double mutants are viable, and exhibit short body length and small brood size compared to N2 and each of the single mutants. The liquid chromatography-tandem mass spectrometry analysis demonstrated that the levels of ADMA and SDMA were abolished in the prmt-1;prmt-5 double mutants. Both prmt-1 and prmt-5 were required for resistance to heat and oxidative stresses, whereas prmt-5 is not involved in lifespan regulation even when prmt-1 is ablated. This mutant strain would be a useful model animal for investigating the role of asymmetric and symmetric arginine dimethylation in vivo.

Asymmetric Arginine Dimethylation Modulates Mitochondrial Energy Metabolism and Homeostasis in Caenorhabditis elegans.

Protein arginine methyltransferase 1 (PRMT-1) catalyzes asymmetric arginine dimethylation on cellular proteins and modulates various aspects of biological processes, such as signal transduction, DNA repair, and transcriptional regulation. We have previously reported that the null mutant of prmt-1 in Caenorhabditis elegans exhibits a slightly shortened life span, but the physiological significance of PRMT-1 remains largely unclear. Here we explored the role of PRMT-1 in mitochondrial function as hinted by a two-dimensional Western blot-based proteomic study. Subcellular fractionation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that PRMT-1 is almost entirely responsible for asymmetric arginine dimethylation on mitochondrial proteins. Importantly, isolated mitochondria from prmt-1 mutants represent compromised ATP synthesis in vitro, and whole-worm respiration in prmt-1 mutants is decreased in vivo Transgenic rescue experiments demonstrate that PRMT-1-dependent asymmetric arginine dimethylation is required to prevent mitochondrial reactive oxygen species (ROS) production, which consequently causes the activation of the mitochondrial unfolded-protein response. Furthermore, the loss of enzymatic activity of prmt-1 induces food avoidance behavior due to mitochondrial dysfunction, but treatment with the antioxidant N-acetylcysteine significantly ameliorates this phenotype. These findings add a new layer of complexity to the posttranslational regulation of mitochondrial function and provide clues for understanding the physiological roles of PRMT-1 in multicellular organisms.

Non-transcriptional Function of FOXO1/DAF-16 Contributes to Translesion DNA Synthesis.

Forkhead box O (FOXO; DAF-16 in nematode) transcription factors activate a program of genes that control stress resistance, metabolism, and lifespan. Given the adverse impact of the stochastic DNA damage on organismal development and ageing, we examined the role of FOXO/DAF-16 in UV-induced DNA-damage response. Knockdown of FOXO1, but not FOXO3a, increases sensitivity to UV irradiation when exposed during S phase, suggesting a contribution of FOXO1 to translesion DNA synthesis (TLS), a replicative bypass of UV-induced DNA lesions. Actually, FOXO1 depletion results in a sustained activation of the ATR-Chk1 signaling and a reduction of PCNA monoubiquitination following UV irradiation. FOXO1 does not alter the expression of TLS-related genes but binds to the protein replication protein A (RPA1) that coats single-stranded DNA and acts as a scaffold for TLS. In Caenorhabditis elegans, daf-16 null mutants show UV-induced retardation in larval development and are rescued by overexpressing DAF-16 mutant lacking transactivation domain, but not substitution mutant unable to interact with RPA-1. Thus, our findings demonstrate that FOXO1/DAF-16 is a functional component in TLS independently of its transactivation activity.