Nuclear RNA-acetylation can be erased by the deacetylase SIRT7

A large number of RNA modifications are known to affect processing and function of rRNA, tRNA and mRNA 1. The N4-acetylcytidine (ac4C) is the only known RNA acetylation event and is known to occur on rRNA, tRNA and mRNA 2,3. RNA modification by acetylation affects a number of biological processes, including translation and RNA stability 2. For a few RNA methyl modifications, a reversible nature has been demonstrated where specific writer proteins deposit the modification and eraser proteins can remove them by oxidative demethylation 4–6. The functionality of RNA modifications is often mediated by interaction with reader proteins that bind dependent on the presence of specific modifications 1. The NAT10 acetyltransferase has been firmly identified as the main writer of acetylation of cytidine ribonucleotides, but so far neither readers nor erasers of ac4C have been identified 2,3. Here we show, that ac4C is bound by the nucleolar protein NOP58 and deacetylated by SIRT7, for the first time demonstrating reversal by another mechanism than oxidative demethylation. NOP58 and SIRT7 are involved in snoRNA function and pre-ribosomal RNA processing 7–10, and using a NAT10 deficient cell line we can show that the reduction in ac4C levels affects both snoRNA sub-nuclear localization and pre-rRNA processing. SIRT7 can deacetylate RNA in vitro and endogenous levels of ac4C on snoRNA increase in a SIRT7 deficient cell line, supporting its endogenous function as an RNA deacetylase. In summary, we identify the first eraser and reader proteins of the RNA modification ac4C, respectively, and suggest an involvement of RNA acetylation in snoRNA function and pre-rRNA processing.

Analysis of disease model iPSCs derived from patients with a novel Fanconi anemia-like IBMFS ADH5/ALDH2 deficiency

We have recently discovered Japanese children with a novel Fanconi anemia-like inherited bone marrow failure syndrome. This disorder is likely caused by the loss of a catabolic system directed toward endogenous formaldehyde, due to biallelic variants in ADH5 combined with a heterozygous ALDH2*2 dominant-negative allele (rs671), which is associated with alcohol-induced Asian flushing. PHA-stimulated lymphocytes from these patients displayed highly increased numbers of spontaneous sister chromatid exchanges (SCEs), reflecting homologous recombination repair of formaldehyde damage. Here we report that, by contrast, patient-derived fibroblasts showed normal levels of SCEs, suggesting that different cell types or conditions generate varying amounts of formaldehyde. To obtain insights about endogenous formaldehyde production and how defects in ADH5/ALDH2 affect human hematopoiesis, we constructed disease model cell lines, including iPS cells (iPSC). We found that ADH5 is the primary defense against formaldehyde, and ALDH2 provides a backup. DNA repair capacity in the ADH5/ALDH2-deficient cell lines can be overwhelmed by exogenous low-dose formaldehyde as indicated by higher levels of DNA damage than FANCD2-deficient cells. Although ADH5/ALDH2-deficient cell lines were healthy and showed stable growth, disease model iPSCs displayed drastically defective cell expansion when stimulated into hematopoietic differentiation in vitro, displaying increased levels of DNA damage. The expansion defect was partially reversed by treatment with a new small molecule termed C1, which is an agonist of ALDH2, thus identifying a potential therapeutic strategy for the patients. We propose that hematopoiesis or lymphocyte blastogenesis may entail formaldehyde generation that necessitates elimination by ADH5/ALDH2 enzymes.

Purine DNA Lesions at Different Oxygen Concentration in DNA Repair-Impaired Human Cells (EUE-siXPA)

Xeroderma Pigmentosum (XP) is a DNA repair disease characterized by nucleotide excision repair (NER) malfunction, leading to photosensitivity and increased incidence of skin malignancies. The role of XP-A in NER pathways has been well studied while discrepancies associated with ROS levels and the role of radical species between normal and deficient XPA cell lines have been observed. Using liquid chromatography tandem mass spectrometry we have determined the four 5’,8-cyclopurines (cPu) lesions (i.e., 5′R-cdG, 5′S-cdG, 5′R-cdA and 5′S-cdA), 8-oxo-dA and 8-oxo-dG in wt (EUE-pBD650) and XPA-deficient (EUE-siXPA) human embryonic epithelial cell lines, under different oxygen tension (hyperoxic 21%, physioxic 5% and hypoxic 1%). The levels of Fe and Cu were also measured. The main findings of our study were: (i) the total amount of cPu (1.82–2.52 lesions/106 nucleotides) is the same order of magnitude as 8-oxo-Pu (3.10–4.11 lesions/106 nucleotides) in both cell types, (ii) the four cPu levels are similar in hyperoxic and physioxic conditions for both wt and deficient cell lines, whereas 8-oxo-Pu increases in all cases, (iii) both wt and deficient cell lines accumulated high levels of cPu under hypoxic compared to physioxic conditions, whereas the 8-oxo-Pu levels show an opposite trend, (iv) the diastereoisomeric ratios 5′R/5′S are independent of oxygen concentration being 0.29 for cdG and 2.69 for cdA for EUE-pBD650 (wt) and 0.32 for cdG and 2.94 for cdA for EUE-siXPA (deficient), (v) in deficient cell lines Fe levels were significantly higher. The data show for the first time the connection of oxygen concentration in cells with different DNA repair ability and the levels of different DNA lesions highlighting the significance of cPu. Membrane lipidomic data at 21% O2 indicated differences in the fatty acid contents between wild type and deficient cells, envisaging functional effects on membranes associated with the different repair capabilities, to be further investigated.