1.
Characterization of RNA Sensing Pathways in Hepatoma Cell Lines and Primary Human Hepatocytes.
Nicolay, W, Moeller, R, Kahl, S, Vondran, FWR, Pietschmann, T, Kunz, S, Gerold, G
Cells. 2021;(11)
Abstract
The liver is targeted by several human pathogenic RNA viruses for viral replication and dissemination; despite this, the extent of innate immune sensing of RNA viruses by human hepatocytes is insufficiently understood to date. In particular, for highly human tropic viruses such as hepatitis C virus, cell culture models are needed to study immune sensing. However, several human hepatoma cell lines have impaired RNA sensing pathways and fail to mimic innate immune responses in the human liver. Here we compare the RNA sensing properties of six human hepatoma cell lines, namely Huh-6, Huh-7, HepG2, HepG2-HFL, Hep3B, and HepaRG, with primary human hepatocytes. We show that primary liver cells sense RNA through retinoic acid-inducible gene I (RIG-I) like receptor (RLR) and Toll-like receptor 3 (TLR3) pathways. Of the tested cell lines, Hep3B cells most closely mimicked the RLR and TLR3 mediated sensing in primary hepatocytes. This was shown by the expression of RLRs and TLR3 as well as the expression and release of bioactive interferon in primary hepatocytes and Hep3B cells. Our work shows that Hep3B cells partially mimic RNA sensing in primary hepatocytes and thus can serve as in vitro model to study innate immunity to RNA viruses in hepatocytes.
2.
Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage.
Sundheim, O, Vågbø, CB, Bjørås, M, Sousa, MM, Talstad, V, Aas, PA, Drabløs, F, Krokan, HE, Tainer, JA, Slupphaug, G
The EMBO journal. 2006;(14):3389-97
Abstract
Methylating agents are ubiquitous in the environment, and central in cancer therapy. The 1-methyladenine and 3-methylcytosine lesions in DNA/RNA contribute to the cytotoxicity of such agents. These lesions are directly reversed by ABH3 (hABH3) in humans and AlkB in Escherichia coli. Here, we report the structure of the hABH3 catalytic core in complex with iron and 2-oxoglutarate (2OG) at 1.5 A resolution and analyse key site-directed mutants. The hABH3 structure reveals the beta-strand jelly-roll fold that coordinates a catalytically active iron centre by a conserved His1-X-Asp/Glu-X(n)-His2 motif. This experimentally establishes hABH3 as a structural member of the Fe(II)/2OG-dependent dioxygenase superfamily, which couples substrate oxidation to conversion of 2OG into succinate and CO2. A positively charged DNA/RNA binding groove indicates a distinct nucleic acid binding conformation different from that predicted in the AlkB structure with three nucleotides. These results uncover previously unassigned key catalytic residues, identify a flexible hairpin involved in nucleotide flipping and ss/ds-DNA discrimination, and reveal self-hydroxylation of an active site leucine that may protect against uncoupled generation of dangerous oxygen radicals.
3.
Telomerase limits the extent of base pairing between template RNA and telomeric DNA.
Förstemann, K, Lingner, J
EMBO reports. 2005;(4):361-6
Abstract
Telomerase is the ribonucleoprotein reverse transcriptase that adds telomeric DNA repeats to the ends of chromosomes. This involves annealing of the telomerase RNA template to the 3' end of the chromosome, reverse transcription of the RNA template by the telomerase reverse transcriptase polypeptide and translocation. Here, we overexpress and partially purify the catalytically active yeast telomerase core in its natural host and probe telomerase RNA base methylation accessibility with dimethyl sulphate in the presence and absence of a DNA substrate and after substrate elongation. The length of the RNA-DNA hybrid is kept constant at seven base pairs after primer binding and elongation. Thus, new base-pair formation at the 3' end of the substrate during elongation coincides with disruption of base-pair interactions at the other side of the template. Presumably, this circumvents the generation of an exceedingly high energy barrier for translocation and dissociation. Our analysis also corroborates recently proposed yeast telomerase RNA secondary structure models.
4.
Covalent binding of leukotriene A4 to DNA and RNA.
Hankin, JA, Jones, DN, Murphy, RC
Chemical research in toxicology. 2003;(4):551-61
Abstract
Leukotriene A(4) (LTA(4)) is a highly reactive electrophilic intermediate formed during the biosynthesis of the lipid mediators leukotriene B(4) and leukotriene C(4). Deoxynucleosides were found to react as nucleophiles with LTA(4) in aqueous solutions as assessed by UV spectroscopy and electrospray ionization mass spectrometry. Aqueous solutions of native DNA and RNA were also found to react with LTA(4) as assessed by mass spectrometric analysis of the constituent nucleosides derived from enzymatic hydrolysis of the nucleic acids. The most abundant adducts were observed for guanine- and adenine-containing deoxynucleosides and nucleosides. At neutral pH, these reactions led to an overall modification of deoxyguanosine/guanosine residues in DNA and RNA at 15 +/- 1 adducts/10(7) bases and 230 +/- 20 adducts/10(7) bases, respectively, determined by quantitative assay using stable isotope-labeled LTA(4)-nucleoside adduct. An estimation of the relative reactivity of LTA(4) with each of the purine and pyrimidine bases in DNA and RNA was carried out by comparisons of the mass spectral ion abundance of the different adducts (LTA(4)-dAdo, LTA(4)-dCyd, LTA(4)-Thd, LTA(4)-Ado, LTA(4)-Cyd, and LTA(4)-Urd) to the ion signal of known amounts of LTA(4)-dGuo and LTA(4)-Guo standards. The data were corrected for different mass spectrometric response factors that were experimentally determined for each adduct product. The structures of the two most abundant LTA(4)-Guo products were determined by NMR, UV spectroscopy, and mass spectrometry to be 5-hydroxy,12-[Guo-N(2)-yl]-6,8,11,14-eicosatetraenoic acid. Stimulation of human neutrophils with calcium ionophore led to the covalent modification of DNA within the cell as determined by mass spectrometric analysis of lipophilic nucleosides obtained after hydrolysis of extracted DNA. These observations, combined with the intracellular site of 5-lipoxygenase translocation and LTA(4) biosynthesis at the nuclear envelope, suggest that LTA(4) may have access to DNA and RNA within cells and furthermore modify nucleic acids in situ following the activation of 5-lipoxygenase and initiation of LTA(4) biosynthesis.