RNA Modification in Archaea and Eukaryotes

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 DESCRIPTION (provided by applicant): Post-transcriptional RNA processing and modifications can regulate gene expression, which is essential for the control of cellular metabolism, growth, and differentiation. Studying these processes in complex systems is often challenging and sometimes not even feasible. Archaea often have eukaryote-like processes, but at a basic level. Therefore, they can serve as much simpler model systems to gain insights into complex eukaryotic events. Broad long-term objectives of this application are to characterize various RNA modification events in Archaea and extend the studies to eukaryotes. Specific aims of this proposal are: (1) Characterization of different structural elements of guide and target RNAs during box C/D and box H/ACA guide RNP-mediated modifications; (2) Determination of the interactions of different box C/D and box H/ACA core proteins with each other and with RNAs during modification reactions; (3) Identification and characterization of the accessory proteins associated with core proteins of archaeal guide sRNPs; and (4) Characterization of 54 in specific eukaryotic tRNAs and determination of the role of Pus10 protein in its synthesis. Various in vitro and in vivo techniques will be used to address the above-mentioned aims. Lead(II) induced cleavage mapping and EMSA of 32P-labeled transcripts will be done using mutant box C/D and H/ACA core proteins for the structural studies of guide and target RNAs during RNP assembly and during catalysis. In vitro modification reactions will be done in parallel with these studies. In vivo interactions of different components of RNPs will be studied by expressing mutant proteins and RNAs in corresponding gene-deleted strains of Haloferax volcanii. In vivo cross-linking of His-tagged core proteins as well as an aptamer-containing box C/D RNA followed by Ni-NTA chromatography of H. volcanii cell extracts will be done to isolate sRNP-associated proteins. Isolated proteins will be identified by mass spectrometry and further characterized after determining their identity. Presence or absence of ¿ in cellular RNAs will be determined by primer extension following CMCT and U-specific reactions, and 2'-O-methylation will be determined by primer extension under limited dNTP concentrations. Extracts of Pus10 knock down HeLa cells will be tested for any reduction in tRNA 54 activity. Expression of a plasmid-borne copy of the Pus10 gene in these cells will be done to attempt rescue of any observed reduced activity. Defects and changes in RNA modification patterns and modification machinery have been observed in diseases and infections. Therefore, our studies of different RNA modification processes will help us understand these processes under normal conditions, and any changes in these processes that may occur under diseased conditions.