Association of RNA with the uracil‐DNA‐degrading factor has major conformational effects and is potentially involved in protein folding

Békési, Angéla; Pukáncsik, Mária; Haasz, Peter; Felfoldi, Lilla; Leveles, Ibolya; Muha, Villő; Hunyadi‐Gulyás, Éva; Erdei, Anna; Medzihradszky, Katalin F.; Vértessy, Beáta G.

doi:10.1111/j.1742-4658.2010.07951.x

Cited by 6 publications

(6 citation statements)

References 80 publications

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“…Furthermore, RNase treatment coupled with agarose gel electrophoresis showed the void volume sample contained RNA (Figure 5B). The behaviour of CNPase in these assays is highly similar to the uracil-DNA-degrading factor [32]. No peak at the void volume is observed for the catalytic domain (data not shown), which indicates the N-terminal domain of CNPase is involved in RNA binding, at least in the case of E. coli RNA.…”

Section: Resultsmentioning

confidence: 76%

Myelin 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase: Active-Site Ligand Binding and Molecular Conformation

et al. 2012

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The 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) is a highly abundant membrane-associated enzyme in the myelin sheath of the vertebrate nervous system. CNPase is a member of the 2H phosphoesterase family and catalyzes the formation of 2′-nucleotide products from 2′,3′-cyclic substrates; however, its physiological substrate and function remain unknown. It is likely that CNPase participates in RNA metabolism in the myelinating cell. We solved crystal structures of the phosphodiesterase domain of mouse CNPase, showing the binding mode of nucleotide ligands in the active site. The binding mode of the product 2′-AMP provides a detailed view of the reaction mechanism. Comparisons of CNPase crystal structures highlight flexible loops, which could play roles in substrate recognition; large differences in the active-site vicinity are observed when comparing more distant members of the 2H family. We also studied the full-length CNPase, showing its N-terminal domain is involved in RNA binding and dimerization. Our results provide a detailed picture of the CNPase active site during its catalytic cycle, and suggest a specific function for the previously uncharacterized N-terminal domain.

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Section: Resultsmentioning

confidence: 76%

Myelin 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase: Active-Site Ligand Binding and Molecular Conformation

et al. 2012

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“…First, similarly to the case of hypermethylated DNA, uracil–DNA may show a decreased response and interaction with transcriptional regulators, activators or other morphogenetic factors required specifically during pupal metamorphosis. Second, one or more factor(s), functional only in the pupal stage, may process uracil–DNA resulting in genome instability and defects in cell cycle progression or cell death [47], [48]. Beyond UNG that is missing from Drosophila , other uracil–DNA glycosylases would be suspect for this role.…”

Section: Resultsmentioning

confidence: 99%

Uracil-Containing DNA in Drosophila: Stability, Stage-Specific Accumulation, and Developmental Involvement

et al. 2012

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Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil–DNA glycosylase and dUTPase. Lack of the major uracil–DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200–2,000 uracil/million bases, quantified using a novel real-time PCR–based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil–DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil–DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil–DNA in this evolutionary clade.

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“…The behaviour of PNK/CPDase proteins in these assays was highly similar to the uracil-DNA degrading factor and mouse CNPase [27, 49]. CNPase has also been shown to interact with RNA in vitro and co-purify with poly(A) + RNA; the catalytic domain of CNPase has been shown to be sufficient for binding with single stranded RNA homopolymers [50, 51].…”

Section: Resultsmentioning

confidence: 99%

Structural similarities and functional differences clarify evolutionary relationships between tRNA healing enzymes and the myelin enzyme CNPase

et al. 2017

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BackgroundEukaryotic tRNA splicing is an essential process in the transformation of a primary tRNA transcript into a mature functional tRNA molecule. 5′-phosphate ligation involves two steps: a healing reaction catalyzed by polynucleotide kinase (PNK) in association with cyclic phosphodiesterase (CPDase), and a sealing reaction catalyzed by an RNA ligase. The enzymes that catalyze tRNA healing in yeast and higher eukaryotes are homologous to the members of the 2H phosphoesterase superfamily, in particular to the vertebrate myelin enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase).ResultsWe employed different biophysical and biochemical methods to elucidate the overall structural and functional features of the tRNA healing enzymes yeast Trl1 PNK/CPDase and lancelet PNK/CPDase and compared them with vertebrate CNPase. The yeast and the lancelet enzymes have cyclic phosphodiesterase and polynucleotide kinase activity, while vertebrate CNPase lacks PNK activity. In addition, we also show that the healing enzymes are structurally similar to the vertebrate CNPase by applying synchrotron radiation circular dichroism spectroscopy and small-angle X-ray scattering.ConclusionsWe provide a structural analysis of the tRNA healing enzyme PNK and CPDase domains together. Our results support evolution of vertebrate CNPase from tRNA healing enzymes with a loss of function at its N-terminal PNK-like domain.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-017-0084-2) contains supplementary material, which is available to authorized users.

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Association of RNA with the uracil‐DNA‐degrading factor has major conformational effects and is potentially involved in protein folding

Cited by 6 publications

References 80 publications

Myelin 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase: Active-Site Ligand Binding and Molecular Conformation

Myelin 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase: Active-Site Ligand Binding and Molecular Conformation

Uracil-Containing DNA in Drosophila: Stability, Stage-Specific Accumulation, and Developmental Involvement

Structural similarities and functional differences clarify evolutionary relationships between tRNA healing enzymes and the myelin enzyme CNPase

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