Investigating the function of snR30 during ribosome biogenesis
AbstractsnR30 is an essential and atypical H/ACA box guide RNA conserved across all eukaryotes. In general, the H/ACA ribonucleic protein (RNP) complex is formed by Cbf5, Gar1, Nop10, and Nhp2 associating with an H/ACA guide RNA. This complex is typically involved in the conversion of uridine to pseudouridine. The snR30 RNP, however, is essential for processing of precursor ribosomal RNA (rRNA) at three sites. These cleavages are required for production of mature 18S rRNA which is necessary for ribosome biogenesis. snR30 differs from other H/ACA guide RNAs in that the binding of the target RNA occurs in the lower half of the unpaired pocket within the terminal hairpin compared to traditional H/ACA RNAs. Additional proteins including Utp23, Utp24, Rrp7, Rok1, and others interact either with snR30 or snR30-interacting proteins. This project aims at reconstituting and characterizing a purified snR30 RNP complex to study the most critical function of H/ACA RNPs during ribosome biogenesis.
The H/ACA core protein complex Cbf5-Nop10-Gar1 was purified using glutathione- and Nickel-sepharose chromatography. Synthesis and purification of RNA was achieved through a series of different techniques. First, purified yeast genomic DNA was used for the amplification of snR30 and 35S rRNA. Sequences were cloned into a plasmid. Next, PCR products of these sequences were used as templates in in vitro transcription to generate RNA. Finally, short target RNA comprising a fragment of rRNA was purified using anion exchange chromatography while snR30 was purified by size exclusion chromatography. The target RNA was fluorescently labeled with a fluorescein dye at the oxidized 3’ end. With these components, it now becomes possible to study how snR30 interacts with the H/ACA proteins and how the snR30 RNP complex binds to and dissociates from target RNA, e.g by determining the affinity for rRNA.