Accumulation of H/ACA snoRNPs depends on the integrity of the conserved central domain of the RNA-binding protein Nhp2p

Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes, How box H/ACA snoRNPs a re assembled remains unknown. Here we show that yeast Nhp2p, a core compone nt of these particles, directly binds RNA. In vitro, Nhp2p interacts with h igh affinity with RNAs containing irregular stem-loop structures but shows weak affinity for poly(A), poly(C) or for double-stranded RNAs, The central region of Nhp2p is believed to function as an RNA-binding domain, since it is related to motifs found in various RNA-binding proteins. Removal of two amino acids that shortens a putative beta -strand element within Nhp2p cen tral domain impairs the ability of the protein to interact with H/ACA snoRN As in cell extracts. In vivo, this deletion prevents cell viability and lea ds to a strong defect in the accumulation of H/ACA snoRNAs and Gar1p, These data suggest that proper direct binding of Nhp2p to H/ACA snoRNAs is requi red for the assembly of H/ACA snoRNPs and hence for the stability of some o f their components. In addition, we show that converting a highly conserved glycine residue (G(59)) within Nhp2p central domain to glutamate significa ntly reduces cell growth at 30 and 37 degreesC, Remarkably, this modificati on affects the steady-state levels of H/ACA snoRNAs and the strength of Nhp 2p association with these RNAs to varying degrees, depending on the nature of the H/ACA snoRNA, Finally, we show that the modified Nhp2p protein whose interaction with H/ACA snoRNAs is impaired cannot accumulate in the nucleo lus, suggesting that only the assembled H/ACA snoRNP particles can be effic iently retained in the nucleolus.