To bind or not to bind: Characterization of binding interactions between X29 and U8snoRNA [abstract]

Abstract only availableFaculty Mentor: Brenda Peculis, BiochemistryU8-snoRNP is involved in the processing of the 5.8S and the 28S rRNA, both of which are needed for the formation of the large ribosomal subunit (Peculis and Steitz, 1991). A nucleolar protein, dubbed X29, has the ability to bind and decap U8snoRNA, giving it the capacity to degrade U8RNA (Tomasevic and Peculis, 1999; Ghosh et al, 2004). Initially found in Xenopus, X29 is evolutionarily conserved in vertebrates from humans to sea squirts (MT, unpublished). The x-ray crystallography structure of X29 shows the protein can exist in the form of a homodimer (Scarsdale et al, 2006). The goal of this project was to determine whether the homodimer form or the monomeric version of X29 binds U8RNA and is catalytically active, as well as identify the protein:protein and protein:RNA contacts. I used chemical crosslinkers and identified a 60kD putative crosslink in X29, and formation of a 60kD band was also identified with the human homologue, H29K, but this forms with a lower efficiency. 4-thio-U mediated RNA (UV) crosslinking was used to identify the binding sites for X29 on U8RNA. All crosslinking assays were performed with mutant or truncated RNAs and proteins to more precisely map sites of interaction. The data from X29 were compared to that of H29K to determine whether the protein's activities were conserved among or differed between species. The results of these experiments showed that X29 and H29K both show abilities to form dimers and crosslink to U8 though to differing efficiencies. The U8 mutants have identified putative interaction sites between U8RNA and the proteins, which we are in the process of mapping more precisely

Binding properties of X29 protein and RNA

Abstract only availableThe Peculis Lab previously demonstrated that X29 protein binds U8 snoRNA with high affinity and specificity in Xenopus Laevis [1]. U8 snoRNA is a C/D box RNP required for pre-rRNA maturation of 5.8s and 28s rRNA in the nucleolus. X29 is a Nudix hydrolase that decaps the U8 RNA at the m7G and m227G caps [2]. Together these two components may interact in vivo to regulate the rate of ribosome biogenesis and thus, the rates of cell growth and cell division. My work has focused on characterizing the interaction between these two molecules and I have been working on two interrelated projects. The first project involves generating mutations to alter one amino acid in the protein sequence. Based on crystal structure data, the amino acid tryptophan, at position F49, lies in the putative RNA binding site on the X29 protein. The mutation will substitute a tryptophan for the 'wild type' phenylalanine residue at this position. After the mutagenesis, the protein is expressed in bacteria in BL21 cells and the mutated protein is purified. We predict the protein would contain a fluorescence property such that when analyzed by flourimitry we will be able to determine RNA binding and possibly address stoichiometry and dimer formation. The second project involves cross-linking wild type X29 protein to U8 RNA and mapping the cross-link on the RNA. Cross-linking reactions followed by reverse transcription using 5' end-labeled oligo DNA primers will identify 'stops' which are UV- and protein-dependent. We will be able to map the precise nucleotide on the RNA and interpret this in the framework of the proposed secondary structure for U8 snoRNA. The results of these experiments will greatly aid the research of the X29 protein and its binding capabilities to RNA. References: 1. Tomasevic, N. And B. Peculis, Identification of a U8 snoRNA-specific binding protein. J Biol Chem, 1999. 274: p. 35914-20. 2. Ghosh, T., et al., Xenopus U8 snoRNA binding protein is a conserved nuclear decapping enzyme. Mol Cell, 2004. 13: p. 817- 828.NSF-REU Program in Biological Sciences & Biochemistr

Exploring the functional decapping ability of the dogfish shark Nudt16 homolog

Abstract only availableA nuclear decapping and binding protein, X29/Nudt16, was originally characterized in Xenopus, and has also been characterized in humans. It is suggested that not only the protein's sequence, but also its functions has been preserved through evolution. The purpose of this study is to characterize a homologue of Nudt16 in the dogfish shark, which on an evolutionary scale is quite diverged from frogs and mammals. Here the open reading frame encoding the dogfish shark protein was amplified via PCR and cloned into an expression vector. When placed into bacteria under the proper growth conditions, a Histidine-tagged dogfish shark protein is synthesized. The protein can be purified from bacteria. The purified dogfish shark protein will be tested to determine if it has the same biochemical properties as the mammalian and frog proteins: can the protein decap RNA in vitro and can it bind RNA directly. Does this protein have catalytic activity that is essential for a healthy life?NSF grant to B. Peculus, NSF-REU Program in Biological Sciences & Biochemistr