Identification of novel proteins associated with yeast snR30 small nucleolar RNA (original) (raw)

H/ACA small nucleolar RNPs (snoRNPs) that guide pseudouridylation reactions are comprised of one small nucleolar RNA (snoRNA) and four common proteins (Cbf5, Gar1, Nhp2 and Nop10). Unlike other H/ACA snoRNPs, snR30 is essential for the early processing reactions that lead to the production of 18S ribosomal RNA in the yeast Saccharomyces cerevisiae. To determine whether snR30 RNP contains specific proteins that contribute to its unique functional properties, we devised an affinity purification strategy using TAP-tagged Gar1 and an RNA aptamer inserted in snR30 snoRNA to selectively purify the RNP. Northern blotting and pCp labeling experiments showed that S1-tagged snR30 snoRNA can be selectively purified with streptavidin beads. Protein analysis revealed that aptamer-tagged snR30 RNA was associated with the four H/ACA proteins and a number of additional proteins: Nop6, ribosomal proteins S9 and S18 and histones H2B and H4. Using antibodies raised against Nop6 we show that endogenous...