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

• Published in: The FEBS journal 2011, Vol.278 (2), p.295-315
• Main Authors: Bekesi, Angela, Pukancsik, Maria, Haasz, Peter, Felfoldi, Lilla, Leveles, Ibolya, Muha, Villo, Hunyadi-Gulyas, Eva, Erdei, Anna, Medzihradszky, Katalin F, Vertessy, Beata G
• Format: Article
• Language: English
• Subjects: Animals; Biochemistry; Circular Dichroism; conformational states; cotranslational folding; Deoxyribonucleic acid; DNA; DNA - metabolism; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Drosophila melanogaster - chemistry; Drosophila melanogaster - metabolism; Drosophila Proteins - chemistry; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Electrophoretic Mobility Shift Assay; Endopeptidases - metabolism; Fluorescent Dyes - chemistry; Genetic recombination; Hydrophobic and Hydrophilic Interactions; Insects; Models, Molecular; Oligodeoxyribonucleotides - metabolism; Oligoribonucleotides - metabolism; Protein Binding - physiology; Protein Conformation; Protein Folding; Protein Structure, Secondary; Protein Unfolding; Pupa - chemistry; Pupa - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Ribonucleases - analysis; Ribonucleases - metabolism; RNA - metabolism; RNA binding; RNA, Double-Stranded - metabolism; RNA, Messenger - analysis; RNA, Messenger - metabolism; RNA, Ribosomal, 16S - analysis; RNA, Ribosomal, 16S - metabolism; RNA, Ribosomal, 23S - analysis; RNA, Ribosomal, 23S - metabolism; RNA‐assisted folding; Spectrometry, Fluorescence; Surface Properties; Temperature; Transition Temperature; uracil‐DNA‐degrading factor
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2010.07951.x

Recently, a novel uracil-DNA-degrading factor protein (UDE) was identified in Drosophila melanogaster, with homologues only in pupating insects. Its unique uracil-DNA-degrading activity and a potential domain organization pattern have been described. UDE seems to be the first representative of a new protein family with unique enzyme activity that has a putative role in insect development. In addition, UDE may also serve as potential tool in molecular biological applications. Owing to lack of homology with other proteins with known structure and/or function, de novo data are required for a detailed characterization of UDE structure and function. Here, experimental evidence is provided that recombinant protein is present in two distinct conformers. One of these contains a significant amount of RNA strongly bound to the protein, influencing its conformation. Detailed biophysical characterization of the two distinct conformational states (termed UDE and RNA-UDE) revealed essential differences. UDE cannot be converted into RNA-UDE by addition of the same RNA, implying putatively joint processes of RNA binding and protein folding in this conformational species. By real-time PCR and sequencing after random cloning, the bound RNA pool was shown to consist of UDE mRNA and the two ribosomal RNAs, also suggesting cotranslational RNA-assisted folding. This finding, on the one hand, might open a way to obtain a conformationally homogeneous UDE preparation, promoting successful crystallization; on the other hand, it might imply a further molecular function of the protein. In fact, RNA-dependent complexation of UDE was also demonstrated in a fruit fly pupal extract, suggesting physiological relevance of RNA binding of this DNA-processing enzyme.